Normal Response Research Articles

A new mutation of Sgms1 causes gradual hearing loss associated with a reduced endocochlear potential

Sgms1 encodes sphingomyelin synthase 1, an enzyme in the sphingosine-1-phosphate signalling pathway, and was previously reported to underlie hearing impairment in the mouse. A new mouse allele, Sgms1tm1a, unexpectedly showed normal Auditory Brainstem Response thresholds. We found that the Sgms1tm1a mutation led to incomplete knockdown of transcript to 20% of normal values, which was enough to support normal hearing. The Sgms1tm1b allele was generated by knocking out exon 7, leading to a complete lack of detectable transcript in the inner ear. Sgms1tm1b homozygotes showed largely normal auditory brainstem response thresholds at first, followed by progressive loss of sensitivity until they showed severe impairment at 6 months old. The endocochlear potential was consistently reduced in Sgms1tm1b mutants at 3, 4 and 8 weeks old, to around 80mV compared with around 120mV in control littermates. The stria vascularis showed a characteristic irregularity of marginal cell surfaces and patchy loss of Kcnq1 expression at their apical membrane, and expression analysis of the lateral wall suggested that marginal cells were the most likely initial site of dysfunction in the mutants. Finally, significant association of auditory thresholds with DNA markers within and close to the human SGMS1 gene were found in the 1958 Birth Cohort, suggesting that SGMS1 variants may play a role in the range of hearing abilities in the human population.

Synaptic Plasticity in the Injured Brain Depends on the Temporal Pattern of Stimulation.

Neurostimulation protocols are increasingly used as therapeutic interventions, including for brain injury. In addition to the direct activation of neurons, these stimulation protocols are also likely to have downstream effects on those neurons' synaptic outputs. It is well known that alterations in the strength of synaptic connections (long-term potentiation, LTP; long-term depression, LTD) are sensitive to the frequency of stimulation used for induction; however, little is known about the contribution of the temporal pattern of stimulation to the downstream synaptic plasticity that may be induced by neurostimulation in the injured brain. We explored interactions of the temporal pattern and frequency of neurostimulation in the normal cerebral cortex and after mild traumatic brain injury (mTBI), to inform therapies to strengthen or weaken neural circuits in injured brains, as well as to better understand the role of these factors in normal brain plasticity. Whole-cell (WC) patch-clamp recordings of evoked postsynaptic potentials in individual neurons, as well as field potential (FP) recordings, were made from layer 2/3 of visual cortex in response to stimulation of layer 4, in acute slices from control (naive), sham operated, and mTBI rats. We compared synaptic plasticity induced by different stimulation protocols, each consisting of a specific frequency (1 Hz, 10 Hz, or 100 Hz), continuity (continuous or discontinuous), and temporal pattern (perfectly regular, slightly irregular, or highly irregular). At the individual neuron level, dramatic differences in plasticity outcome occurred when the highly irregular stimulation protocol was used at 1 Hz or 10 Hz, producing an overall LTD in controls and shams, but a robust overall LTP after mTBI. Consistent with the individual neuron results, the plasticity outcomes for simultaneous FP recordings were similar, indicative of our results generalizing to a larger scale synaptic network than can be sampled by individual WC recordings alone. In addition to the differences in plasticity outcome between control (naive or sham) and injured brains, the dynamics of the changes in synaptic responses that developed during stimulation were predictive of the final plasticity outcome. Our results demonstrate that the temporal pattern of stimulation plays a role in the polarity and magnitude of synaptic plasticity induced in the cerebral cortex while highlighting differences between normal and injured brain responses. Moreover, these results may be useful for optimization of neurostimulation therapies to treat mTBI and other brain disorders, in addition to providing new insights into downstream plasticity signaling mechanisms in the normal brain.

Diagnosis of hypoprolactinemia.

Prolactin is a polypeptide hormone composed of 199 amino acids, synthesized by lactotroph cells. Its primary effects are on the mammary gland and gonadal axes, but it also influences different organs and systems, particularly metabolic functions. Current literature has mainly focused on the diagnosis, monitoring, and treatment of hyperprolactinemia. Due to the lack of a well-established effective treatment for hypoprolactinemia, it is not clinically emphasized. Therefore, data on its diagnosis is limited. Hypoprolactinemia has been associated with metabolic dysfunctions such as type 2 diabetes mellitus, fatty liver, dyslipidemia, fertility problems, sexual dysfunction, and increased cardiovascular disease. While often seen as a part of combined hormone deficiencies due to pituitary damage, isolated prolactin deficiency is rare. Hypoprolactinemia can serve as a marker for extensive pituitary gland damage and dysfunction.Low or undetectable serum prolactin levels and the absence of a sufficient prolactin peak in the thyrotropin-releasing hormone (TRH) stimulation test are considered diagnostic for hypoprolactinemia. Gender appears to influence both basal prolactin levels and TRH stimulation test responses. Basal prolactin levels of, at least, 5 ng/mL for males and 7 ng/mL for females can be used as cut-off levels for normal prolactin reserve. Minimum peak prolactin responses of 18 ng/mL for males and 41 ng/mL for females to TRH stimulation can exclude hypoprolactinemia. However, larger population studies across different age groups and sexes are needed to better define normal basal prolactin levels and prolactin responses to the TRH stimulation test.

Evaluation of the follicular fluid thiol/disulfide balance among patients with poor ovarian response.

This study aimed to compare the thiol/disulfide balance, myeloperoxidase, and ischemia-modified albumin levels in the follicular fluid (FF) of poor ovarian response (POR) and normal ovarian response (NOR) women who received intracytoplasmic sperm injection (ICSI). The study was performed between March 2021 and April 2022 at the Department of Obstetrics and Gynecology, Center for Reproductive Medicine, Ankara City Hospital. The study included 27 POR and 35 NOR women who underwent ICSI. FF was obtained after the controlled ovarian stimulation cycle. The FF thiol/disulfide balance was detected using spectrophotometric methods. A correlation analysis was conducted to determine whether these oxidative stress markers could contribute to predicting oocyte quality. Disulfide levels were significantly higher in the NOR group than in the POR group (p=0.014). The number of fertilized egg (2PN) oocytes was positively correlated with the total thiol level (r=0.258, p=0.046). The disulfide level was positively correlated with the anti-Müllerian hormone level (r=0.262, p=0.039) and the total number of retrieved oocytes (r=0.335, p=0.008). The disulfide levels differed significantly between the NOR and POR groups. The statistically significant differences of fewer metaphase II oocytes and lower percentage of good-quality embryos in the NOR group compared to the POR group might have resulted from the NOR group's elevated disulfide levels. The total thiol levels correlated with the total of 2PN oocytes. Future studies should examine the thiol/disulfide balance at assisted reproductive technology centers to predict which oocytes could be fertilized.

Estimation of the prevalence of isolated inter-scalenic compression from simultaneous arterial and venous photoplethysmography in patients referred for suspected thoracic outlet syndrome.

In patients with suspected thoracic outlet syndrome (TOS), diagnosing inter-scalenic compression could lead to minimally invasive treatments. During photo-plethysmography, completing a 30-sec 90° abduction, external rotation ("surrender" position) by addition of a 15-sec 90° antepulsion "prayer" position, allows quantitative bilateral analysis of both arterial (A-PPG) and venous (V-PPG) results. We aimed at determining the proportion of isolated arterial compression with photo-plethysmography in TOS-suspected patients. &#xD;Approach: We studied 116 subjects recruited over 4 months (43.3+/-11.8 years old, 69% females). Fingertip A-PPG and forearm V-PPG were recorded on both sides at 125 Hz and 4 Hz respectively. A-PPG was converted to PPG amplitude and expressed as percentage of resting amplitude (%rest). V-PPG was expressed as percentage of the maximal value (%max) observed during the "Surrender-Prayer" maneuver. Impairment of arterial inflow during the surrender (As+) or prayer (Ap+) phases were defined as a pulse-amplitude either < 5%rest, or < 25%rest. Incomplete venous emptying during the surrender (Vs+) or prayer (Vp+) phases were defined as V-PPG values either <70%max, or <87%max. &#xD;Main results: Of the 16 possible associations of encodings, As-Vs-Ap-Vp- was the most frequent observation assumed to be a normal response. Isolated arterial inflow without venous outflow (As+Vs-) impairment in the surrender position was observed in 10.3% [95%CI: 6.7-15.0%] to 15.1% [95%CI: 10.7 - 20.4%] of limbs. &#xD;Significance: Simultaneous A-PPG and V-PPG can discriminate arterial from venous compression and then potentially inter-scalenic from other levels of compressions. As such, it opens new perspectives in evaluation and treatment of TOS.&#xD.

Haploinsufficiency of the Parkinson’s disease gene synaptojanin1 is associated with abnormal responses to psychomotor stimulants and mesolimbic dopamine signaling

The synaptojanin-1 (SYNJ1) gene is known to be important for dopamine-related disorders. Recent evidence has demonstrated that Synj1 deficient mice (Synj1+/−) have impairments in dopaminergic synaptic vesicular recycling. However, less is known about how Synj1 deficits affect the mesolimbic system, reward processing, and motivated behavior. To examine the role of the Synj1 gene in motivated behavior, we subjected male and female Synj1+/− and Synj1+/+ mice to a battery of behavioral tests evaluating hedonic responses, effortful responding, and responses to psychomotor stimulants. We observed that Synj1+/− mice exhibit few differences in reward processing and motivated behavior, with normal hedonic responses and motivated responding for sucrose. However, male but not female Synj1+/− demonstrated an attenuated conditioned place preference for cocaine that could not be attributed to deficits in spatial memory. To further understand the dopamine signaling underlying the attenuated response to cocaine in these mutant mice, we recorded nucleus accumbens dopamine in response to cocaine and observed that Synj1+/− male and female mice took longer to reach peak dopamine release following experimenter-administered cocaine. However, female mice also showed slower decay in accumbens dopamine that appear to be linked to differences in cocaine-induced DAT responses. These findings demonstrate that SYNJ1 deficiencies result in abnormal mesolimbic DA signaling which has not previously been demonstrated. Our work also highlights the need to develop targeted therapeutics capable of restoring deficits in DAT function, which may be effective for reversing the pathologies associated with Synj1 mutations.

P-564 Decoding ovarian response: the predictive power of FSH receptor gene expression in controlled ovarian stimulation

Abstract Study question Can profiling of variation in follicle-stimulating hormone receptor (FSHR) gene expression forecast response to Controlled Ovarian Stimulation (COS) in Assisted Reproductive Techniques? Summary answer Full-length FSHR transcript was the most common mRNA species. Nine distinct transcripts were characterised, but only one (deletion Exon 6) was associated with altered response. What is known already AMH levels and age are considered useful predictors of ovarian response to COS (high AMH and young age typically associated with high yields of oocytes; older ages and low AMH associated with fewer oocytes). However, it is not uncommon to see patients who do not fit this pattern, highlighting the limitations of these parameters in predicting response. Age-independent biomarkers capable of revealing patients at elevated risk of hyperstimulation, or those who might produce fewer than the optimal number of oocytes, are desirable. Alternative transcripts of the FSHR gene have been proposed as an explanation for variation in response to COS. Study design, size, duration FSHR splice variants in mRNA in granulosa cells from 126 patients were examined utilising long-read nanopore sequencing. This strategy permitted the sequencing of mRNA variants without fragmentation, allowing the detection of all splicing events in each mRNA strand. In addition to being categorised based on age and AMH levels, patients were also classified by the expression of FSHR transcript variants and their response to stimulation (high, low, or normal response). Participants/materials, setting, methods Patterns of FSHR mRNA splice variants were analysed in each of 126 patients. This involved extraction of RNA from the granulosa cells, followed by long-read nanopore sequencing, permitting analysis of whole transcripts in a single, contiguous sequence ‘read’. Patients were classified based upon their level of response to COS (low, normal, high), taking into account their age, AMH level and the number of oocytes retrieved. Patterns of FSHR transcript variation were compared to COS response. Main results and the role of chance Full-length FSHR transcript was detected in 97.6% of patients and skipping of Exon 6 was observed in 68%. Five novel transcripts were discovered, including partial insertion of ∼100bp segments of introns 1 or 2, seen in 6.55% of samples. In total, nine distinct FSHR mRNA variants were detected. As expected, increasing age was associated with higher FSH requirement per oocyte collected (p = 0.018), and AMH levels declined with age (2.8± 1.42 in women &amp;lt;38 versus 1.58 ±1.29 in women ≥38; p = 0.011). Consistent with previous analyses, AMH levels and amount of FSH per oocyte retrieved were negatively correlated (p &amp;lt; 0.001). One-third of the patients (33%) in this cohort displayed responses to COS that were not considered to be consistent with expectations based upon age and AMH levels. Expression of the novel FSHR transcript variants was not associated with FSH needed per oocyte retrieved (p = 0.78), nor the proportion of mature oocytes (p = 0.95).The only transcript potentially linked to an altered response to COS was the previously reported mRNA species, lacking exon 6, associated with significantly greater numbers of oocytes produced (p = 0.0087). Interestingly, women ≥38 expressed novel variants more frequently than their younger counterparts (p = 0.018), potentially indicative of an age-related decline in transcriptional fidelity. Limitations, reasons for caution It is possible that the use of controlled ovarian stimulation might alter the dynamics of transcription in cumulus cells. Therefore, it is possible that the spectrum of transcripts seen in this study might differ from those present in unstimulated cycles. Wider implications of the findings Expected associations between age, AMH levels and response to COS were observed. Skipping exon 6 may be associated with a superior response to COS, suggesting FSHR mRNA profiling might have a role in tailoring controlled ovarian stimulation protocols. Novel transcript variants were detected and seen most often in older women. Trial registration number not applicable

P-406 Late paternal effect rooted in sperm DNA double-strand breaks (DSBs) correlates with the likelihood of miscarriage in patients with poor ovarian response

Abstract Study question Can paternal risk factors be minimized and prevented in ICSI procedure? Summary answer The influence of DSBs can be prevented in ICSI cases with normal ovarian response, whereas DSB is exclusively associated with miscarriage in poor ovarian reserve. What is known already Sperm DNA fragmentation (SDF) is intricately linked to male fertility capacity, with a high prevalence affecting 20% of men with unexplained infertility and up to 50% of those with idiopathic infertility. Despite these statistics, the association between paternal DNA damages and clinical outcomes is controversial. Variations in SDF effects may be influenced by female factors associated with maternal DNA repair activity. To elucidate this interrelationship, we assessed SDF effects on diverse ovarian responses using POSEIDON stratification. The goal was to identify potential paternal risk factors for ART as a preventative solution and devising preventative strategies. Study design, size, duration This retrospective study included 323 couples who underwent ICSI cycles at Lee Women’s Hospital. Normal responders (n = 89) were identified based on female age (&amp;lt;35 years) and AMH levels (≥1.2 ng/mL). Patients (n = 234) fulfilling POSEIDON group 1–4 criteria were categorized as poor ovarian response (POR). Clinical outcomes assessed included clinical pregnancy, miscarriage, and live birth rates. Participants/materials, setting, methods Paternal risk factors include male age, semen parameters (CEROS II CASA, Hamilton-Thorne), and two types of SDF evaluation. LensHooke® R10 kit (Bonraybio, Taiwan) was used to detect total SDF (tSDF), which includes single-strand breaks and double-strand breaks (DSBs). LensHooke® R11 kit was specifically used for DSB detection. Kruskal-Wallis test was conducted to compare controls and cases of ART failure. Univariate and multivariate logistic regression analyses were employed to identify potential factors. Main results and the role of chance We sought to evaluate paternal effects during the early gestation stage by comparing parental characteristics in groups with clinical pregnancy and those without pregnancy. No significant differences in paternal indications were observed between the groups. Notably, significantly lower AMH levels and advanced maternal age were prevalent in pregnancy failures across both normal responders and POR cases, highlighting maternal factors’ dominance in these outcomes. When dissecting paternal effects during the later gestation stage, male factors did not show a significant difference between the group with live birth and the group with miscarriage among normal responders. Nevertheless, several paternal pathogenic indications were identified between the groups in POR. Miscarriages were associated with compromised sperm total motility (68% vs. 54.5%, p = 0.01), reduced normal morphology rate (5.5% vs. 2.5%, p = 0.03), and elevated DSB levels (7.5% vs. 18.8%, p = 0.01). Furthermore, multivariate regression analysis identified high DSBs as an exclusive paternal factor in miscarriage risk (Odds ratio: 1.19, 95% confidence interval 1.04–1.36, p = 0.01). In the ROC curve, a DSBs threshold of 19%, based on the R11 technique, offers significant value in predicting miscarriage with an accuracy of 81%. Limitations, reasons for caution The limitations of the current study are single-center trial, and relatively small sample size. Further clinical trials are needed to robustly establish the relationship between DSB and miscarriage. Wider implications of the findings The impact of DSBs is overlooked in normal responders but not in POR due to different levels of maternal DNA repair activity. These results advocates for (1) the necessity of DSBs (R11) assessment for POR, and (2) egg donation as a potential strategy for couples facing high DSBs and POR. Trial registration number CS2-20012

O-196 Men who fathered embryos with poor pre-implantation development rates exhibit an increased frequency of sperm with high DNA accessibility

Abstract Study question Do differing levels of DNA accessibility in sperm affect the ability of embryos generated by ART to undergo pre-implantation development? Summary answer Individuals whose sperm generated embryos that failed to efficiently form blastocysts tend to have more sperm with high levels of DNA accessibility. What is known already Chromatin is a complex composed of nucleic acids and proteins that serves to condense and regulate the genome in all eukaryotic cells. Sperm chromatin is packaged into a structure with higher density than that found in precursor germ cells or somatic cells. Samples from individuals with decreased normal semen parameters have previously been shown to possess an increased frequency of sperm with aberrant chromatin packaging. However, existing methodologies for measuring sperm chromatin packaging are indirect and non-quantitative. Study design, size, duration Prospective cohort study, registered January 2020. We adapted an enzymatic methodology for measuring DNA accessibility known as NicE-view (Nicking Endonuclease-mediated visualization), for use in human sperm. DNA accessibility was quantified in three millions of individual sperm from semen obtained from 60 individuals. Both total sperm and sperm selected for motility via swim-up were analyzed for each participant. Participants/materials, setting, methods Participants sought treatment for infertility at Universitätsspital Basel and possessed normospermic levels in conventional semen analysis. Participant partners had normal endocrine parameters and ovulation response to hormonal stimulation. 20 couples obtained pregnancies without the need for ART, while 40 individuals who were treated using ART and divided into two groups differing in pre-implantation development rates: (1) 20 with blastocyst development of &amp;gt; 50% of all zygotes and (2) 20 produced 0-1 blastocyst following embryo culture. Main results and the role of chance We quantified DNA accessibility and the presence of single stranded DNA nicks in more than 3 million sperm derived from 117 samples. We found that while DNA nicks were only present in a small fraction of the sperm analyzed, DNA accessibility levels varied dramatically with the data indicating a bimodal distribution. At a single sperm level, DNA accessibility and the presence of single stranded DNA damage both correlated with nuclear size. We found that DNA nicking was more abundant in total sperm samples compared to sperm selected for motility via swim-up preparation. In contrast, sperm with high levels of DNA accessibility were more abundant following swim-up, perhaps resulting from capacitation. We observed that individuals with a history of low blastocyst development were more likely to have high frequencies of sperm harboring DNA nicks and high DNA accessibility. We also measured increased frequencies of sperm with high DNA accessibility to negatively correlate with conventional semen parameter values. Limitations, reasons for caution Our study cohort consists of only 60 individuals. Variation is also observed between individuals within each participant category, so further analysis with increased participant numbers is required to strengthen these conclusions. Wider implications of the findings Our results suggest measuring DNA accessibility in sperm from normospermic individuals provide additional information about the efficiency of pre-implantation development following ART. Future work examining the sources of increased DNA accessibility in sperm will also be interesting to identify possible treatments for such individuals. Trial registration number SNCTP000003654 | NCT04256668

P-771 Does follicle size impact effect of follicular flushing on oocyte retrieval rate and embryology outcomes?

Abstract Study question Is the effect of follicular flushing on oocyte retrieval rate (RR) and embryology outcome influenced by follicular size? Summary answer Flushing improved RR in discordant growth with smaller(8-12 mm) follicles and larger(&amp;gt;22mm) follicle. Flushing did not impact embryological outcomes adversely. It increased top quality embryo What is known already Follicular flushing is done in poor responder for improving retrieval rate. There are very few studies in normal or hyper responders. Studies show controversial results of flushing, some showing benefits, while others, a detrimental effect because of increased intra-follicular pressure, increased procedure time, and change in paracrine milieu of oocytes by dilution with flushing medium No study has addressed whether increased retrieval rates with flushing are dependent on follicular size and indirectly maturity of oocyte. Most studies flushed with fixed volume of 1-2ml as size of follicle was not taken into account, making volume inadequate for completely flushing larger follicles. Study design, size, duration Prospective cohort study conducted from September 2022 to August 2023. Group 1: Study group- follicles were flushed after aspiration. Total patients were 120. They were divided into 3 groups according to follicular size and fluid volume aspirated with sample size calculated as A:306 small follicles 8-12 mm(&amp;lt;1.0ml), B:467 medium follicles 13-22 mm (1.0-6.0ml) C:102 large follicle &amp;gt; 22 mm (&amp;gt;6.0 ml). Group 2- Control group where follicles aspirated without flushing in 280 patients, 2572 follicles. Participants/materials, setting, methods Ethical approval taken. Inclusion - patients 21-40years of age undergoing oocyte retrieval for IVF/ICSI cycle with antagonist protocol Exclusion of oocyte of indeterminate origin ie one aspirate have two oocytes Group1 After aspiration of follicle with double lumen oocyte retrieval needle, follicle flushed with media volume equivalent to follicular volume. Group2 only follicular aspiration and volume measured Analysis done using SPSS-25 version. Primary outcome retrieval rate(RR) of oocytes. Secondary outcome: fertilization rate, percentage of top quality embryos(TQE)on day3&amp;5, blastocyst rate. Main results and the role of chance Flushing follicle significantly increased RR in smaller follicles(8-12mm) compared to controls 159/311 (51.12%)vs 176/780 (22.56%) p = &amp;lt;0.001 and also in larger follicles(&amp;gt;6ml) 76/109 (69.72%)vs 116/219 (52.96%) p = &amp;lt;0.01 No difference seen in optimum size follicles (13-22mm) 337/470 (71.7%)vs 1099/1573(69.86%) p = 0.44. Overall, there was statistically significant increase in RR with flushing 567/890 (63.7%)vs 1391/2572 (54.08%) p = &amp;lt;0.001. Significantly more time was taken for flushing vs control groups 16.50vs 12.15 minutes p = &amp;lt;0.001. Secondary outcomes like fertilization rate was not different with flushing 476/567 (83.95±15.01%)vs 1125/1391(80.88±19.01 %) p = 0.21. Percentage of TQE (day 3) were significantly more in flushing group 321/476 (67.43±25.41%)vs control 642/1125 (57.07±25.94%)p = &amp;lt;0.01. TQE (day 5) were significantly more in flushing group 142/476 (29.83±11.02)vs control 251/1125 (22.31±12.11%)p=0.04. Blastocyst rate remained similar in both groups 196/476 (41.18±16.73 %) vs 403/1125 ( 35.82±13.90 %) p = 0.12. Subgroup analysis showed that younger women (&amp;lt;35yrs)had a significantly higher RR with flushing 66.33±16.29%vs controls 57.88±13.68% (p = &amp;lt;0.01), compared to older women &amp;gt;35 years 60.29±16.37%vs 56.35±17.30% (p = 0.38). Women with higher AMH had better RR with flushing 63.71±15.11%vs controls 56.50±14.33 (p = 0.01) compared to women with lower AMH (&amp;lt;1 ng/ml) where flushing made no difference 63.47±19.59%vs 60.62±14.93(p = 0.79). Normal and hyper responders had significantly higher RR with flushing 66.16±15.11vs controls 57.53±13.85%(p = &amp;lt;0.001) compared to poor responders (&amp;lt;5 oocytes) 51.07±18.07vs 56.24±27.47(p = 0.55). Limitations, reasons for caution Although RR was better with flushing in small and large follicles we could not translate the advantage to pregnancy rate(PR) PR could not be calculated as some patients wanted 2 embryos transferred. Since patient had one each embryos from small and optimum follicles both were transferred. Further RCT required. Wider implications of the findings Practice of follicular flushing needs re-evaluation Women with discordant growth should be taken for flushing as it increases RR in small and large follicles Role of flushing should be widened beyond poor responders to younger women, with normal or hyper response increasing oocyte yield which may increase cumulative pregnancy rate Trial registration number not applicable

Immune modulation permits tolerance and engraftment in a murine model of late-gestation transplantation

AbstractIn utero hematopoietic cell transplantation is an experimental nonmyeloablative therapy with potential applications in hematologic disorders, including sickle cell disease (SCD). Its clinical utility has been limited due to the early acquisition of T-cell immunity beginning at ∼14 weeks gestation, posing significant technical challenges and excluding treatment fetuses evaluated after the first trimester. Using murine neonatal transplantation at 20 days postcoitum (DPC) as a model for late-gestation transplantation (LGT) in humans, we investigated whether immune modulation with anti-CD3 monoclonal antibody (mAb) could achieve donor–specific tolerance and sustained allogeneic engraftment comparable with that of early-gestation fetal recipient at 14 DPC. In allogeneic wild-type strain combinations, administration of anti-CD3 mAb with transplantation resulted in transient T-cell depletion (TCD) followed by central tolerance induction confirmed by donor–specific clonal deletion and skin graft tolerance. Normal immune responses to the third-party major histocompatibility complex and viral pathogens were preserved, and graft-versus-host disease did not occur. We further demonstrated the successful application of this approach in the Townes mouse model of SCD. These findings confirm the developing fetal T-cell response as a barrier to LGT and support transient TCD as a safe and effective immunomodulatory strategy to overcome it.

Comparative pathogenesis of three Mayaro virus genotypes in the cynomolgus macaque.

Mayaro virus (MAYV), a mosquito-borne alphavirus, is considered an emerging threat to public health with epidemic potential. Phylogenetic studies show the existence of three MAYV genotypes. In this study, we provide a preliminary analysis of the pathogenesis of all three MAYV genotypes in cynomolgus macaques (Macaca facicularis, Mauritian origin). Significant MAYV-specific RNAemia and viremia were detected during acute infection in animals challenged intravenously with the three MAYV genotypes, and strong neutralizing antibody responses were observed. MAYV RNA was detected at high levels in lymphoid tissues, joint muscle and synovia over 1 month after infection, suggesting that this model could serve as a promising tool in studying MAYV-induced chronic arthralgia, which can persist for years. Significant leucopenia was observed across all MAYV genotypes, peaking with RNAemia. Notable differences in the severity of acute RNAemia and composition of cytokine responses were observed among the three MAYV genotypes. Our model showed no outward signs of clinical disease, but several major endpoints for future MAYV pathology and intervention studies are described. Disruptions to normal blood cell counts and cytokine responses were markedly distinct from those observed in macaque models of CHIKV infection, underlining the importance of developing non-human primate models specific to MAYV infection.

A cylindrical shell model with distributed springs for a rotating tire

In a companion paper (Vol. 248, Article 108227), we demonstrated the dependence of tire parameters on the modal characteristics of a stationary tire. For a more realistic scenario, when tire rotation is considered, the modal characteristics are veered due to centrifugal, Coriolis, and gyroscopic effects. In this paper, we study the forced vibration response of a rotating tire and also consider the effect of static load on modal characteristics. The tire tread is modeled as a cylindrical shell and the sidewall is modelled using distributed springs in axial, circumferential, and radial directions. The tire inflation pressure is accounted for, which generates pre-stresses in the tire belt in circumferential and axial directions. The material model incorporates an orthotropic composite structure of the tire with multiple stacked layers of steel belt reinforcing. The governing equations of motion of the rotating shell are derived and further reduced to the corresponding eigenvalue problem. Galerkin’s projection with orthogonal basis functions is used to obtain the natural frequencies and associated mode shapes. For the forced vibration, the tire is excited by a harmonic point load at a fixed location, and the response is calculated by superimposing the basis functions of the free vibration problem. The analytical model predictions are compared against the computation finite element (FE) model developed in ABAQUS® and experimental modal testing results. To understand the effects of rotation on wave propagation, dispersion relations were obtained, and the wave number spectrum of the rotating tire was compared with that of the stationary one. Finally, to understand the dependence of natural frequencies on tire rotation and normal load, Campbell diagrams and frequency response functions (FRFs) were obtained from the proposed model. The model shows a fairly good agreement with experimental results and FE simulations.

A Study on the Effect of Housing on Physiological Responses of Nellore Lambs Kasala

A study was carried out to find out the effect of housing on physiological responses of Nellore Jodipi lambs under tropical environmental conditions. Thirty weaned Nellore ram lambs of 3-4 months age were selected based on body weight (average initial body weight) and randomly divided into three groups of ten animals each. The lambs in Group-I were housed with morum flooring and asbestos roofing with tree shade, while Group-II lambs were housed with morum flooring and asbestos roofing without tree shade and Group-III lambs were housed with elevated slatted flooring and galvalume roofing without tree shade. Temperature and humidity values were significantly (P&lt;0.05) different among all the groups. Lower mean maximum temperature (°C) values were observed in group III (35.53±0.28) when compared to group I (36.22±0.27) and II (37.29±0.25) and the lower average maximum relative humidity (%) was 69.59±2.92 in group III followed by group II (69.88±2.94) and I (70.65±2.96). The mean physiological responses viz., rectal temperature, respiration rate and pulse rate were significantly (P&lt;0.05) lower in group III (102.38±1.00°F, 26.25±1.00bpm, 69.07±1.00beats/min.) when compared to group I (102.60±1.00°F, 31.60±1.00bpm, 82.10±1.00beats/min.) and II (102.85±1.00°F, 33.56±1.00bpm, 87.08±1.00beats/min.), respectively. It was concluded that the housing system of elevated slatted flooring with galvalume roofing proved to be effective in controlling heat stress and maintaining normal physiological responses for better growth performance of Nellore ram lambs.

Development of an Ex Vivo Functional Assay for Prediction of Irradiation Related Toxicity in Healthy Oral Mucosa Tissue.

Radiotherapy in the head-and-neck area is one of the main curative treatment options. However, this comes at the cost of varying levels of normal tissue toxicity, affecting up to 80% of patients. Mucositis can cause pain, weight loss and treatment delays, leading to worse outcomes and a decreased quality of life. Therefore, there is an urgent need for an approach to predicting normal mucosal responses in patients prior to treatment. We here describe an assay to detect irradiation responses in healthy oral mucosa tissue. Mucosa specimens from the oral cavity were obtained after surgical resection, cut into thin slices, irradiated and cultured for three days. Seven samples were irradiated with X-ray, and three additional samples were irradiated with both X-ray and protons. Healthy oral mucosa tissue slices maintained normal morphology and viability for three days. We measured a dose-dependent response to X-ray irradiation and compared X-ray and proton irradiation in the same mucosa sample using standardized automated image analysis. Furthermore, increased levels of inflammation-inducing factors-major drivers of mucositis development-could be detected after irradiation. This model can be utilized for investigating mechanistic aspects of mucositis development and can be developed into an assay to predict radiation-induced toxicity in normal mucosa.

Decreased Extracellular Vesicle Vasorin in Severe Preeclampsia Plasma Mediates Endothelial Dysfunction.

Preeclampsia (PE) is a serious pregnancy complication affecting 5-8% of pregnancies globally. It is a leading cause of maternal and neonatal morbidity and mortality. Despite its prevalence, the underlying mechanisms of PE remain unclear. This study aimed to determine the potential role of vasorin (VASN) in PE pathogenesis by investigating its levels in extracellular vesicles (EV) and its effects on vascular function. We conducted unbiased proteomics on urine-derived EV from severe PE (sPE) and normotensive pregnant women (NTP), identifying differential protein abundances. Out of one hundred and twenty proteins with ≥ ±1.5-fold regulation at P<0.05 between sPE and NTP, we focused on Vasorin (VASN), which is downregulated in sPE in urinary EV, in plasma EV and in the placenta and is a known regulator of vascular function. We generated EV with high VASN content from both human and murine placenta explants (Plex EV), which recapitulated disease-state-dependent effects on vascular function observed when treating murine aorta rings (MAR) or human aortic endothelial cells (HAEC) with murine or human plasma-derived EV. In normal murine pregnancy, VASN increases with gestational age (GA), and VASN is decreased in plasma EV, in placenta tissue and in Plex EV after intravenous administration of adenovirus encoding short FMS-like tyrosine kinase 1 (sFLT-1), a murine model of PE (murine-PE). VASN is decreased in plasma EV, in placenta tissue and in EV isolated from conditioned media collected from placenta explants (Plex EV) in patients with sPE as compared to NTP. Human sPE and murine-PE plasma EV and Plex EV impair migration, tube formation, and induces apoptosis in human aortic endothelial cells (HAEC) and inhibit acetylcholine-induced vasorelaxation in murine vascular rings (MAR). VASN over-expression counteracts the effects of sPE EV treatment in HAEC and MAR. RNA sequencing revealed that over-expression or knock down of VASN in HAEC results in contrasting effects on transcript levels of hundreds of genes associated with vasculogenesis, endothelial cell proliferation, migration and apoptosis. The data suggest that VASN, delivered to the endothelium via EV, regulates vascular function and that the loss of EV VASN may be one of the mechanistic drivers of PE. What is NewVASN in circulating plasma EV in sPE is reduced compared with VASN content in plasma EV of gestational age-matched pregnant women.VASN is encapsulated and transported in EV and plays a pro-angiogenic role during pregnancy.VASN should be explored both for its pro-angiogenic mechanistic role and as a novel biomarker and potential predictive diagnostic marker for the onset and severity of PE.What Are the Clinical Implications?VASN plays a role in maintaining vascular health and the normal adaptive cardiovascular response in pregnancy. A decrease of VASN is observed in sPE patients contributing to cardiovascular maladaptation.Strategies to boost diminished VASN levels and/or to pharmacologically manipulate mechanisms downstream of VASN may be explored for potential therapeutic benefit in PE.The decrease in EV-associated VASN could potentially be used as a (predictive) biomarker for PE.

Natural variation in infection specificity of Caenorhabditis briggsae isolates by two RNA viruses.

Antagonistic relationships such as host-virus interactions potentially lead to rapid evolution and specificity in interactions. The Orsay virus is so far the only horizontal virus naturally infecting the nematode C. elegans. In contrast, several related RNA viruses infect its congener C. briggsae, including Santeuil (SANTV) and Le Blanc (LEBV) viruses. Here we focus on the host's intraspecific variation in sensitivity to these two intestinal viruses. Many temperate-origin C. briggsae strains, including JU1264 and JU1498, are sensitive to both, while many tropical strains, such as AF16, are resistant to both. Interestingly, some C. briggsae strains exhibit a specific resistance, such as the HK104 strain, specifically resistant to LEBV. The viral sensitivity pattern matches the strains' geographic and genomic relationships. The heavily infected strains mount a seemingly normal small RNA response that is insufficient to suppress viral infection, while the resistant strains show no small RNA response, suggesting an early block in viral entry or replication. We use a genetic approach from the host side to map genomic regions participating in viral resistance polymorphisms. Using Advanced Intercrossed Recombinant Inbred Lines (RILs) between virus-resistant AF16 and SANTV-sensitive HK104, we detect Quantitative Trait Loci (QTLs) on chromosomes IV and III. Building RILs between virus-sensitive JU1498 and LEBV-resistant HK104 followed by bulk segregant analysis, we identify a chromosome II QTL. In both cases, further introgressions of the regions confirmed the QTLs. This diversity provides an avenue for studying virus entry, replication, and exit mechanisms, as well as host-virus specificity and the host response to a specific virus infection.

Detection of CLCF1 protein expression by flow cytometry

Cardiotrophin-like cytokine factor 1 (CLCF1) is an IL-6 family cytokine with neurotrophic and immuno-modulating functions. CLCF1 mRNA has been detected in primary and secondary lymphoid organs, and up-regulation of CLCF1 mRNA levels has been associated with the T helper (Th) 17 polarization. However, information regarding CLCF1 expression by immune cells at the protein level remains scarce. We have developed a methodology that uses a monoclonal antibody (mAb) directed against CLCF1 for the detection of human and mouse CLCF1 by flow cytometry. We have successfully detected CLCF1 protein expression in cells from the mouse pro-B cell line Ba/F3 that were transduced with CLCF1 cDNA. Interestingly, we found that the anti-CLCF1 mAb inhibits CLCF1 biological activity in vitro by binding to an epitope that encompasses site III of the cytokine. Moreover, we have detected CLCF1 expression in mouse splenic T cells, as well as in vitro differentiated Th1 cells. The specificity of the fluorescence signal was demonstrated using Clcf1-deficient lymphocytes generated using a conditional knock-out mouse model. The detection of CLCF1 protein by flow cytometry will be a valuable tool to study CLCF1 expression during normal and pathological immune responses.

Exploring ligands that target von Willebrand factor selectively under oxidizing conditions through docking and molecular dynamics simulations.

The blood protein von Willebrand factor (VWF) is a large multimeric protein that, when activated, binds to blood platelets, tethering them to the site of vascular injury and initiating blood coagulation. This process is critical for the normal hemostatic response, but especially under inflammatory conditions, it is thought to be a major player in pathological thrombus formation. For this reason, VWF has been the target for the development of anti-thrombotic therapeutics. However, it is challenging to prevent pathological thrombus formation while still allowing normal physiological blood coagulation, as currently available anti-thrombotic therapeutics are known to cause unwanted bleeding, in particular intracranial hemorrhage. This work explores the possibility of inhibiting VWF selectively under the inflammatory conditions present during pathological thrombus formation. In particular, the A2 domain of VWF is known to inhibit the neighboring A1 domain from binding to the platelet surface receptor GpIbα, and this auto-inhibitory mechanism has been shown to be removed by oxidizing agents released during inflammation. Hence, finding drug molecules that bind at the interface between A1 and A2 only under oxidizing conditions could restore such an auto-inhibitory mechanism. Here, by using a combination of computational docking, molecular dynamics simulations, and free energy perturbation calculations, a ligand from the ZINC15 database was identified that binds at the A1A2 interface, with the interaction being stronger under oxidizing conditions. The results provide a framework for the discovery of drug molecules that bind to a protein selectively in the presence of inflammatory conditions.

Transcutaneous vagal nerve stimulation during lower body negative pressure

BackgroundPara-sympathetic vagal activation has profound influence on heart rate and other cardiovascular parameters. We tested the hypothesis that transcutaneous Vagal Nerve Stimulation (tVNS) through the auricular branch of the vagus nerve would attenuate the normal sympathetic response to central blood volume reduction by lower body negative pressure (LBNP). Method10 healthy volunteers (6 female; age 21 ± 2 years; weight 62 ± 13 kg; height 167 ± 12 cm) were included in this cross-over design trial. After 15 min rest in supine position, subjects underwent three 15-min periods of 30 mmHg LBNP intervention with and without cyclic tVNS stimulation. Continuous cardiovascular parameters (Nexfin) were recorded. ResultsOverall tVNS did not convincingly attenuate sympathetic response to central hypovolemia. Deactivation of the tVNS during LBNP resulted in increased MAP at 2.3 ± 0.5 mmHg (P < 0.001). Comparing the cyclic actual active stimulation periods to periods with pause during tVNS intervention showed a decrease in HR by 72.9 ± 11.2 to 70.2 ± 11.6 bpm (mean ± SD; P < 0.05), and concomitant increases in SV (86.0 ± 12.1 to 87.2 ± 12.6 mL; P < 0.05), MAP (82.9 ± 6.3 to 84.0 ± 6.2 mmHg; P < 0.05) and TPR (1116.0 ± 111.1 to 1153 ± 104.8 dyn*s/cm5; P < 0.05). ConclusiontVNS in 30 s cycles during LBNP can selectively attenuate HR, prompting a compensatory augmented sympathetic response. It would appear the method used in this study at least, has an isolated cardiac inhibitory effect probably mediated by augmented vagal activity on the sinoatrial or atrio-ventricular node, possibly in combination with reduced activity in the sympathetic cardiac nerve.