Day 1 Research Articles

Prevalence of mosaicism in day 5 vs. day 6 human blastocysts

Background and Aim: Chromosomal mosaicism is defined by having two or more distinct cell lines present within an embryo, as a consequence of mitotic errors. In this study, we investigate the prevalence of mosaicism in day 5 (D5) versus day 6 (D6) blastocysts.

AMD3100 PROMOTES A RAPID AND EFFICIENT MOBILIZATION OF BONE MARROW-DERIVED HEMATOPOIETIC STEM CELLS IN THE BLOODSTREAM OF BALB/C MICE

Hematopoietic stem cells (HSCs) are the basis of bone marrow (BM) transplantation. Its clinical success depends on the efficient HSCs mobilization from BM to peripheral blood (PB) for leukapheresis and clinical harnessing. Although granulocyte-colony stimulating factor (G-CSF) is the “gold standard” to stimulate the HSCs trafficking to PB, AMD3100 has emerged as a well-tolerated pharmacological strategy without significant toxicity. Evaluate the influence of AMD3100 and G-CSF on HSCs mobilization in BM to PB in BALB/c mice. Forty-five BALB/c mice were distributed into 5 groups to receive pre-treatment with intraperitoneal (i.p.) injection of phosphate-buffered saline (PBS) solution (160 μl/i.p.) or G-CSF (200 μg/kg/i.p.) for 4 consecutive days. On day 5 (D5), mice were also injected with PBS or AMD3100 (5 mg/kg/i.p.). These mice were then grouped into PBS+PBS control group (CG, n = 9) PBS+G-CSF group (n = 9), PBS+AMD3100 group (n = 9), G-CSF+PBS group (n = 9) and G-CSF+AMD3100 group (n = 9). After 1 hour, BM and PB samples were subsequently collected. The mobilized CD45 + CD34 + HSCs were assessed ex vivo using Flow Cytometry. Statistical analyses were performed through GraphPad Prism (v.5) using the One-way ANOVA test with Tukey's post-test. Mice of the PBS+AMD3100 group exhibited significant HSCs enrichment into the BM and mobilization to PB when compared to CG and to the PBS+G-CSF group ( p =0.0002). Furthermore, it was observed that mice of the G-CSF+PBS group also exhibited a higher frequency of HSCs in BM and PB compared to CG and to the PBS+G-CSF group ( p =0.0002). Interestingly, mice of the G-CSF+PBS group had a significant increase of HSCs in BM when compared to CG and to the PBS+G-CSF group ( p =0.0002). In contrast, mice of the G-CSF+AMD3100 group had the highest rate of HSCs mobilization in PB when compared to CG and to the PBS+G-CSF group ( p =0.0002). The pre-treatment with G-CSF for 4 consecutive days or just a single dose of AMD3100 on D5 induced a higher enrichment of HSCs in BM and PB. However, the combination of pre-treatment with G-CSF with a single dose of AMD3100 on D5 just induced the highest mobilization of HSCs in PB. These data demonstrate that AMD3100 induces more rapid and robust mobilization of CD34+ HSCs in the BM and PB, unlike G-CSF, which acts in a dose-dependent manner. FAPEAM, CAPES and CNPq.

GRANULOCYTE-COLONY STIMULATING FACTOR (G-CSF) ENHANCES THE MOBILIZATION OF BONE MARROW-DERIVED MESENCHYMAL STEM CELLS IN THE PERIPHERAL BLOOD OF BALB/C MICE

Mesenchymal stem cells (MSCs) have emerged in recent years as beneficial tools for cell therapy and autologous/allogeneic transplantation and their clinical harnessing considers the bone marrow (BM) and peripheral blood (PB). However, this requires a process of endogenous mobilization that offers a non-invasive alternative. Thus, granulocyte colony-stimulating factor (G-CSF) and AMD3100 (Plerixafor ® ) are well known and clinically approved to stimulate MSCs mobilization, but data on their potential activity in MSCs mobilization are still scarce. Evaluate the influence of G-CSF and AMD3100 stimulation on MSCs mobilization from BM to PB in BALB/c mice. For this study, 45 male BALB/c mice aged 3-4 weeks were used. They were divided into 5 groups: PBS+PBS control group (CG, n = 9), PBS+GCSF group (n = 9), PBS+AMD3100 group (n = 9), G-CSF+PBS group (n = 9) and G-CSF+AMD3100 group (n = 9). Pre-treatment was performed with intraperitoneal (i.p.) injections of phosphate-buffered saline (PBS, 160 μl/i.p.) or G-CSF (200 μg/kg/i.p.) for 4 consecutive days. On day 5 (D5), 24 hours after the last injection of PBS or G-CSF, mice were also injected with PBS or AMD3100 (5 mg/kg/i.p.). After 1 hour, BM and PB samples were subsequently collected. The mobilized CD14+CD73+CD90+CD105+ MSCs were then evaluated ex vivo using Flow Cytometry. Statistical analyzes were performed through GraphPad Prism (v.5) using One-way ANOVA test with Tukey's post-test. Based on this study, it was found that there were no statistically significant differences for MSCs mobilization in the BM in any of the studied groups; however, the PBS+AMD3100 group had the highest rate of MSCs enrichment within the BM. Notably, mice of the G-CSF+PBS group exhibited the highest rate of MSCs mobilization in the PB when compared to the CG (p < 0.0001), while the G-CSF+AMD3100 group showed no statistically significant difference for MSCs mobilization in PB. Furthermore, the mice of the PBS+G-CSF group and the PBS+AMD3100 group did not exhibit a significant increase in MSCs mobilization in the PB. With these results, it is verified that the pre-treatment with G-CSF is the most suitable to induce the MSCs mobilization to PB in a dose-dependent manner. Our data demonstrated that AMD3100 proved not to be suitable for MSCs mobilization in both BM and PB. FAPEAM, CAPES and CNPq.

THE EFFECT OF GRANULOCYTE COLONY-STIMULATING FACTOR (G-CSF) ADMINISTRATION ON CYTOKINE PRODUCTION BY BONE MARROW AND PERIPHERAL BLOOD DERIVED STEM CELLS IN PRIMARY CULTURE

Granulocyte colony-stimulating factor (G-CSF) is a growth factor that plays an essential role in the modulation of hematopoiesis through direct effects on proliferation, survival, and mobilization of bone marrow (BM) and peripheral blood (PB) derived stem cells (SCs). Evaluate the influence of G-CSF administration on cytokine production by SCs derived from BM and PB in primary culture. SCs were isolated from BM and PB from 27 BALB/c mice pre-treated for 4 consecutive days with intraperitoneal (i.p.) injections of phosphate-buffered saline (PBS) solution (160 μl/i.p.) or G-CSF (200 μg/kg) at 24-hours intervals. On day 5 (D5), mice were also injected with PBS or G-CSF again. SCs were obtained through BM mononuclear cell isolation by Ficoll-Hypaque™gradient and then stimulated or not with G-CSF in cell culture. Cytokines in the supernatant were quantified by flow cytometry - Cytometric Bead Array (CBA) using the BD™Mouse TH1/TH2/TH17 Cytokine Kit according to manufacturer's instructions. The cytokines measured were IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ and IL-17. The kinetics of these cytokines were monitored on days 3, 6, 9, 12 and 15 of culture. FCAP array software (v3.0.1) was used to calculate the concentrations of each cytokine and the mean fluorescence intensity. An increase in IL-2 and IL-4 was observed between days 9 and 12 of BM-derived SCs culture in groups stimulated with G-CSF. In the culture of PB-derived SCs, the increase in IL-2 and IL-4 occurred between days 3 and 9 in cultures stimulated with G-CSF. The production of IL-6 occurred between days 3 and 9 of culture, both in BM- and PB-derived SCs cultures and reduced until day 15 of culture. In non-G-CSF-stimulated BM-derived SCs cultures, IL-6 levels are elevated in relation to cultures that received G-CSF as a stimulus, on days 6 and 9. IL-10 increased in BM- and PB-derived SCs cultures stimulated with G-CSF on day 3, reducing its concentration until day 15. The same event was observed in BM-derived SCs cultures for IFN-γ and TNF-α, which, like IL-10, were elevated in BM-derived SCs cultures. In PB, an increase in TNF-α is observed on day 3, decreasing progressively until day 15. IL-17 increased on day 12 of BM-derived SCs culture, remaining stable on different days in both BM- and PB-derived SCs cultures. G-CSF can modulate or induce the production of different cytokines by SCs derived from BM and PB, contributing to better regulation of immune responses in pathological and therapeutical different contexts. FAPEAM, CAPES and CNPq.

EVALUATION OF ESTRADIOL ON THE HCG TRIGGER DAY IN PREDICTING PREGNANCY AND NEONATAL OUTCOMES OF PATIENTS UNDERGOING IVF/ICSI: A RETROSPECTIVE COHORT STUDY

Aim: Our aim was to investigate the value of serum estradiol (E2) on human chorionic gonadotrophin (hCG) trigger day in predicting pregnancy (ongoing pregnancy with live birth, abortion, iu ex, chemical pregnancy) outcomes and neonatal birth weight.&#x0D; Material-Methods: This retrospective study consisted of two groups: Group 1: Women who had live birth, labor after 22 gestational week (n= 417). Group 2: Women with clinical pregnancy: intrauterine ex, abortus, biochemical and anembryonic pregnancy (n=260). &#x0D; Demographic characteristics (maternal and paternal ages, body mass index (BMI), gravidity, abortion, and living child) were recorded. Clinical indications (male factor, diminished ovarian reserve...), duration of ovulation stimulation, day 3 (D3) E2, follicle stimulating hormone and luteinizing hormone levels, antral follicle count, total doses of stimulating agents, progesteron and estradiol levels on trigger day, number of oocytes retrieved, number of mature oocytes, endometrial thickness on trigger and oocyte pick-up day were compared between the two groups.&#x0D; Binary Logistic Regression, (Backward LR model) was used to determine the factors affecting the risk of not having a live birth.&#x0D; Results: Totally 677 pregnant women were included in the study. We found increased live birth rate in pregnant whom E2 level was higher on the trigger day. There was a statistically significant relation between E2 level on trigger day and newborn weight in live birth (p=0.005) It was determined that the male factor and age of the women are an important parameter that affect not having a live birth (p

Viral loads in clinical samples of men with monkeypox virus infection: a French case series

BackgroundMonkeypox virus (MPXV) is currently spreading among men who have sex with men, outside of sub-Saharan Africa, and close contact during sex seems to be one of the key pathways of viral transmission in the current outbreak. Our aim was to describe the distribution of MPXV in the human body, as it might play a role in its dissemination through sexual contact.MethodsThe study population in this case series consisted of patients with confirmed MPXV infection attending the Pitié-Salpêtrière Hospital (Paris, France), who had been sampled from multiple anatomical sites, including skin, anus, throat, blood, urine, and semen, at diagnosis and 2 weeks later. We compared the proportion of positive samples and MPXV viral loads (given as PCR cycle thresholds [Ct]) between anatomical sites, and between day 0 (D0) and D14.FindingsOverall, 356 samples were collected between May 20 and June 13, 2022, from 50 men with a median age of 34 years (IQR 29–40). 22 (44%) of the 50 men were classified as HIV-negative on day (D)0, and 22 (44%) were living with HIV. At D0, MPXV detection was more frequent from skin (44 [88%] of 50), anus (30 [71%] of 42), and throat (36 [77%] of 47) than from blood (13 [29%] of 45), urine (nine [22%] of 41), or semen (13 [54%] of 24). Viral loads were significantly higher from skin lesions (Ct 19·8) and anal samples (Ct 20·9) than from throat (Ct 27·2), blood (Ct 32·8), urine (31·1), or semen samples (Ct 27·8). When analysing the 107 samples taken from 24 patients at D14, the proportion of positive samples strongly decreased between D0 and D14 at all sites: skin (four [22%] of 18), anus (two [9%] of 22), throat (none of 21), blood (one [5%] of 21), urine (none of 14), and semen (two [9%] of 11).InterpretationThese data contribute to a better understanding of how the virus might spread between sexual partners over a relatively short period of time. High MPXV viral loads from skin and mucosa, including genital and anal sites, suggest that transmission most likely occurs through direct body contact rather than through the respiratory route or contact with body fluids, which should help to refine the prevention messages delivered to individuals most exposed to the virus.FundingNone.

Methylene Blue-Based Combination Therapy with Amodiaquine Prevents Severe Malaria in an Experimental Rodent Model.

Untreated malaria can progress rapidly to severe forms (<24 h). Moreover, resistance to antimalarial drugs is a threat to global efforts to protect people from malaria. Given this, it is clear that new chemotherapy must be developed. We contribute new data about using methylene blue (MB) to cure malaria and cerebral malaria in a combined therapy with common antimalarial drugs, including mefloquine (MQ) and amodiaquine (AQ). A C57BL6/J mouse model was used in an experimental cerebral malaria model. Mice were infected with Plasmodium berghei ANKA on Day 0 (D0) and the treatment started on D3 (nearly 1% parasitaemia) with AQ, MQ or MB alone or in combination with AQ or MQ. AQ, MQ and MB alone were unable to prevent cerebral malaria as part of a late chemotherapy. MB-based combination therapies were efficient even if treatment began at a late stage. We found a significant difference in survival rate (p < 0.0001) between MBAQ and the untreated group, but also with the AQ (p = 0.0024) and MB groups (p < 0.0001). All the infected mice treated with MB in combination with AQ were protected from cerebral malaria. Partial protection was demonstrated with MB associated with MQ. In this group, a significant difference was found between MBMQ and the untreated group (p < 0.0001), MQ (p = 0.0079) and MB (p = 0.0039). MB associated with AQ would be a good candidate for preventing cerebral malaria.

An additional medium renewal of D4 embryo culture improves the concordance of noninvasive chromosome screening with trophectoderm biopsy.

Our research question was to evaluate the chromosome concordance of trophectoderm (TE) biopsy with noninvasive chromosome screening (NICS) using embryo culture medium renewed twice on Day 3 (D3) and Day 4 (D4). In this study, we evaluated 64 cycles with 223 biopsied blastocysts. These were categorized into two groups based on replacing embryo culture medium on D3 (control group) or on D3 and D4 (experimental group). The fundamental characteristics and main outcomes were compared. The concordance rates of NICS results with TE biopsy were determined according to next generation sequencing results. In total, 103 experimental and 120 control embryo cultures were collected, and the euploid status was analyzed using NICS technology. The overall concordance rates with TE biopsy of the experimental and control groups were 0.86 and 0.75, respectively. Statistically significant difference was found between the two groups. An additional medium renewal of the D4 embryo culture can improve the concordance of NICS with TE biopsy.

Developmental potential of different embryos on day 3: a retrospective study

To investigate how different quality of day 3 (D3) embryos affect blastocyst formation and clinical outcomes. This retrospective study analysed 699 patients undergoing assisted reproductive technology (ART) between January 2017 and February 2021. A total of 2517 D3 embryos were transferred to blastocyst medium for extended culture. D3 embryos were divided into five groups. Grade A, 6–10 cells, symmetrical blastomeres and <20% fragmentation; grade B, 6–10 cells, uneven blastomeres and ≥20% fragmentation; grade C, >10 cells, symmetrical blastomeres and <20% fragmentation; grade D, >10 cells, uneven blastomeres and ≥20% fragmentation; grade E, <6 cells. Status of day 5 (D5) and day 6 (D6) blastocysts and the clinical outcomes (blastocyst transfer) of each D3 embryo were recorded. The grade C group showed a higher D5 blastocyst formation rate and a high-quality blastocyst rate than other groups (p<.05). However, the clinical pregnancy rates in the grade A group were higher than other groups (p<.05). Embryos with low speed of development (grade E group) showed considerable clinical outcomes that were still worth investigating. D3 embryos with less fragmentation and ≥6 symmetrical blastomeres revealed a higher developmental potential, while embryos with 6–10 blastomeres showed the ideal clinical outcomes. Impact Statement What is already known on this subject? Accurate embryo evaluation can effectively reflect the developmental potential of different embryos. The number of blastomeres, proportion of fragmentation, and blastomere symmetry are three important and popular morphologic parameters used for evaluating day 3 (D3) embryos. However, in existing reports, combining these three parameters for embryo evaluation often results in different results. This is because different researchers have chosen different criteria for these three parameters. What do the results of this study add? In this retrospective study, we summarised the medical records of our reproductive centre in the past three years, redefined the evaluation method of the D3 embryos, and analysed the corresponding developmental potential and clinical outcomes. We conclude that although the embryonic development potential of grade C embryos (>10 cells, symmetrical blastomeres and/or <20% fragmentation) is relatively good, the results of grade A embryos (6–10 cells, symmetrical blastomeres and/or <20% fragmentation) are better in terms of clinical outcomes. What are the implications of these findings for clinical practice and/or further research? We believe this is meaningful for embryologists to choose embryos for transfer and predict the clinical outcome of IVF cycles.

Association of pre-treatment somatic cell counts with bacteriological cure following diagnosis of intramammary infection

Antibiotic administration is crucial to ensure the health and productivity of dairy cattle. Mastitis is a disease that is typically a result of an intramammary infection (IMI), and antibiotic regimens are implemented to aid in curing IMI. Diagnosis is usually by detection of elevated milk somatic cell counts (SCC) and/or presence of culturable pathogens in the milk. Antibiotic treatment success is associated with the SCC at the time of treatment, though this correlation is still poorly understood. The objective of this project was to evaluate pre-treatment SCC and its association with IMI cure incidence following a standard antibiotic treatment. We hypothesized that pre-treatment SCC would be significantly lower in cases where the IMI ultimately cured compared to cases where the IMI failed to cure. Milk samples were collected aseptically from lactating cow quarters experiencing clinical or subclinical mastitis (n = 52). Clinical mastitis was diagnosed by a trained milking technician and subclinical mastitis was diagnosed at the quarter level as a SCC > 200,000 cells/mL and presence of bacterical growth in milk at time of treatment. After collection of the day 0 (D0) milk samples, the SCC was enumerated, and the milk sample cultured. Intramammary antibiotic therapy Cetftiofur hydrochloride (Spectramast® LC) was administered once/day for 5 days. Post-treatment samples were collected 14 d (D14) and 28 d (D28) later. A bacteriological cure was confirmed when both the D14 and D28 samples were free of culturable pathogens. The overall cure rate was 46.2%. Interestingly, the cure rates of antibiotic therapy decreased as pre-treatment SCC increased. Quarters that experienced bacteriological cure demonstrated a lower pre-treatment SCC (507,041 cells/mL ± 127.86 SEM, P = 0.01) compared to cows that did not cure, which had high pre-treatment SCC (1,640,392 cells/mL ± 333.28 SEM). Quarters that failed to cure had higher SCC values 28 days post-treatment in comparison to quarters that cured (P < 0.001). Future studies should investigate whether we can develop unique SCC-dependent mastitis treatment protocols which increase mastitis cure rates and enhance overall mammary health.

Feasibility of Neovessel Embolization in a Large Animal Model of Tendinopathy: Safety and Efficacy of Various Embolization Agents.

Targeting neovessels in chronic tendinopathies has emerged as a new therapeutic approach and several embolization agents have been reported. The aim of this study was to investigate the feasibility of embolization with different agents in a porcine model of patellar tendinopathy and evaluate their safety and efficacy. Eight 3-month-old male piglets underwent percutaneous injection of collagenase type I to induce patellar tendinopathies (n = 16 tendons). They were divided into four groups (2 piglets, 4 tendons/group): the control group, 50–100 µm microspheres group, 100–300 µm microspheres group, and the Imipenem/Cilastatin (IMP/CS) group. Angiography and embolization were performed for each patellar tendon on day 7 (D7). The neovessels were evaluated visually with an angiography on day 14. The pathological analysis assessed the efficacy (Bonar score, number of neovessels/mm2) and safety (off-target persistent cutaneous ischemic modifications and presence of off-target embolization agents). The technical success was 92%, with a failed embolization for one tendon due to an arterial dissection. Neoangiogenesis was significantly less important in the embolized groups compared to the control group angiographies (p = 0.04) but not with respect to histology (Bonar score p = 0.15, neovessels p = 0.07). Off-target cutaneous embolization was more frequently depicted in the histology of the 50–100 µm microspheres group (p = 0.02). Embolization of this animal model with induced patellar tendinopathy was technically feasible with different agents and allowed assessing the safety and efficacy of neovessel destruction. Particles smaller than 100 µm seemed to be associated with more complications.

Ebola virus infection induces a delayed type I IFN response in bystander cells and the shutdown of key liver genes in human iPSC-derived hepatocytes.

SummaryLiver damage and an exacerbated inflammatory response are hallmarks of Ebola virus (EBOV) infection. Little is known about the intrinsic response to infection in human hepatocytes and their contribution to inflammation. Here, we present an induced pluripotent stem cell (iPSC)-derived hepatocyte-like cell (HLC) platform to define the hepato-intrinsic response to EBOV infection. We used this platform to show robust EBOV infection, with characteristic ultrastructural changes and evidence for viral replication. Transcriptomics analysis revealed a delayed response with minimal early transcriptomic changes, followed by a general downregulation of hepatic function and upregulation of interferon signaling, providing a potential mechanism by which hepatocytes participate in disease severity and liver damage. Using RNA-fluorescence in situ hybridization (FISH), we showed that IFNB1 and CXCL10 were mainly expressed in non-infected bystander cells. We did not observe an inflammatory signature during infection. In conclusion, iPSC-HLCs are an immune competent platform to study responses to EBOV infection.

Perception-action Coupling Impairments During Simulated Military Operational Stress Are Dependent On Time-of-day

Perception-action coupling (i.e., reading and reacting to the environment) allows military personnel to maneuver safely and effectively through their environment. Exposure to military operational stress (e.g., caloric restriction, sleep loss) and around-the-clock operations may compromise perception-action coupling thereby impacting performance and safety. PURPOSE: To examine the effects of simulated military operational stress (SMOS) and time-of-day on perception-action coupling. METHODS: Fifty-seven military personnel (45 M, 12 W; 26.4 ± 5.6 years) completed a 5-day SMOS protocol. On day 0 (D0), D1 and D4, participants had 8-hour sleep opportunities and received 100% estimated caloric need. On D2 and D3, sleep was restricted and disrupted (two 2-hour sleep opportunities from 01:00 - 03:00 and 05:00 - 07:00), and caloric intake was restricted (50% estimated caloric need). Participants completed a tablet-based perception-action coupling task (PACT) on D1 (18:00, 22:00), D2 (04:00, 18:00, 22:00) and D3 (04:00, 18:00, 22:00). PACT requires participants make perceptual judgements about whether a series of virtual balls fits through virtual apertures of varying sizes. Outcomes of interest included response time (RT), lapses, and incorrect responses. Effects of day X time, day and time were examined using linear mixed models with post hoc pairwise comparisons (Tukey correction). RESULTS: Significant day X time effects were found for RT and lapses (both p < .01) and a significant effect of time was found for incorrect responses (p = .01). RT at 04:00 increased (got slower) by 2.2% across days, but decreased (got faster) by 6.8 and 3.5% across days at 18:00 and 22:00. Lapses at 04:00 increased across days but did not differ from D1 to D3 at 18:00 and 22:00. Participants made 39.3 and 6.0% more incorrect responses at 04:00 than at 18:00 and 22:00 respectively. CONCLUSIONS: Perception-action coupling performance was resilient to SMOS at 18:00 and 22:00, but deteriorated at 04:00. Reduced alertness (lapses), inaccurate perceptual judgements (incorrect responses), and slower response execution (RT) all contributed to perception-action coupling deficits at 04:00 which may reflect impaired performance capabilities of military personnel operating around-the-clock in the field. Support: DoD #W81XWH-17-2-0070

Epidemiological characteristics of P. vivax asymptomatic infections in the Peruvian Amazon.

IntroductionHerein, we tested the hypothesis that Asymptomatic P. vivax (Pv) infected individuals (Asym) feature different epidemiological, clinical and biochemical characteristics, as well as hematological parameters, potentially predictive of clinical immunity in comparison to symptomatic Pv infected individuals (Sym).MethodologyBetween 2018 - 2021, we conducted 11 population screenings (PS, Day 0 (D0)) in 13 different riverine communities around Iquitos city, in the Peruvian Amazon, to identify Pv Sym and Asym individuals. A group of these individuals agreed to participate in a nested case - control study to evaluate biochemical and hematological parameters. Pv Asym individuals did not present common malaria symptoms (fever, headache, and chills), had a positive/negative microscopy result, a positive qPCR result, reported no history of antimalarial treatment during the last month, and were followed-up weekly until Day 21 (D21). Control individuals, had a negative malaria microscopy and qPCR result, no history of antimalarial treatment or malaria infections during the last three years, and no history of comorbidities or chronic infections.ResultsFrom the 2159 individuals screened during PS, data revealed a low but heterogeneous Pv prevalence across the communities (11.4%), where most infections were Asym (66.7%) and submicroscopic (82.9%). A total of 29 Asym, 49 Sym, and 30 control individuals participated in the nested case - control study (n=78). Ten of the individuals that were initially Asym at D0, experienced malaria symptoms during follow up and therefore, were included in the Sym group. 29 individuals remained Asym throughout all follow-ups. High levels of eosinophils were found in Asym individuals in comparison to Sym and controls.ConclusionFor the first-time, key epidemiological, hematological, and biochemical features are reported from Pv Asym infections from the Peruvian Amazon. These results should be considered for the design and reshaping of malaria control measures as the country moves toward malaria elimination.

Comparing the effect of estradiol benzoate and 17β-estradiol plus progesterone on follicular turnover and development, and pregnancy outcomes in a timed artificial insemination protocol

The objective of this study was to compare the efficiency of estradiol benzoate (EB) and 17β-estradiol (E2) associated with progesterone (P4) in a fixed-time artificial insemination (FTAI) protocol. We hypothesized that E2+P4 induces an earlier emergence of a new follicular wave (NFW), improving pre-ovulatory follicle diameter and pregnancy rates to FTAI (P/FTAI). In Exp.1, on Day 0 (D0), all Bos indicus cows (n = 12/group) received an intravaginal P4 device and a dose of PGF2α analogue. On D0, females were randomly assigned to receive EB or E2+P4. On D8.5, P4 intravaginal devices were removed and a dose of PGF2α and EB were administered in all females followed by fixed-timed AI on D10. Between D0 and D10, the dominant follicular growth was determined by ovary ultrasonography exams. On D8.5 and D10 the percentage of color power-Doppler signals in the dominant follicular wall was evaluated. In Exp. 2, 467 females (2-year-old nulliparous [n = 76], primiparous [n = 92] and pluriparous [n = 299]) were subjected to the similar FTAI and assigned to be treated with EB (n = 243) or E2+P4 (n = 224). Pregnancy diagnosis was performed 30 days after FTAI by ultrasonography. The day to emergence of NFW was similar between treatments (EB: 3.7 ± 0.37 vs. E2+P4: 3.3 ± 0.3, P = 0.76). Females treated with E2+P4 presented greater (P = 0.06) follicular growth between the emergence and D9 (1.18 ± 0.07) than those treated with EB (0.97 ± 0.08). There was also a positive effect (P < 0.05) of E2+P4 on diameter of the dominant follicle on D9 (13.0 ± 0.6 vs. 10.9 ± 0.55) and blood perfusion of the follicle wall on D8.5 (49 vs. 40%). There was a treatment by parity category interaction effect on P/FTAI (P < 0.05). Treatment with E2+P4 was advantageous to P/FTAI of primiparous cows (E2+P4: 58% and EB: 30%). However, for nulliparous and pluriparous cows, P/FTAI was similar between treatments (∼50%). In conclusion, in a E2/P4-based protocol for FTAI, E2+P4 is as efficient as EB in inducing new follicular emergence within a similar day range, but it results similar or greater P/FTAI.

Serum lactate and acute mesenteric ischaemia: An observational, controlled multicentre study

BackgroundEarly diagnosis and prompt management of acute mesenteric ischaemia (AMI) are key to survival but remain extremely difficult, due to vague and non-specific symptoms. Serum lactate (SL) is commonly presented as a useful biomarker for the diagnosis or prognosis of AMI. The aim of our study was test SL (1) as a diagnostic marker and (2) as a prognostic marker for AMI. Study designThis was an ancillary multicentre case-control study. Patients with AMI at intensive care unit (ICU) admission were included (AMI group) and matched to ICU patients without AMI (control group). SL was measured and compared on day 0 (D0) and day 1 (D1). Diagnosis and prognosis accuracy were assessed by receiver operating characteristic (ROC) and their area under the curve (AUC). ResultsEach group consisted of 137 matched ICU patients. There was no significant difference of SL between the two groups at D0 or at D1 (p = 0.26 and p = 0.29 respectively). SL was a poor marker of AMI: at D0 and D1, AUC were respectively 0.57 [0.51; 0.63] and 0.60 [0.53; 0.67]. SL at D0 and D1 correctly predicted ICU mortality, independently of AMI (AUC D0: 0.69 [0.59; 0.79] vs. 0.74 [0.65; 0.82]; p = 0.51 and D1: 0.74 [0.64; 0.84] vs. 0.76 [0.66; 0.87]; p = 0.77, respectively, for control and AMI groups]. ConclusionsSL has no specific link with AMI, both for diagnosis and prognosis. SL should not be used for the diagnosis of AMI but, despite its lack of specificity, it may help to assess severity.

The influence of paternal overweight on sperm chromatin integrity, fertilization rate and pregnancy outcome among males attending fertility clinic for IVF/ICSI treatment

BackgroundLow and middle-income countries are facing a rapid increase in obesity and overweight burden, particularly in urban settings. Being overweight in men is associated with infertility and a higher risk to have a low sperm count or no sperm in their ejaculate. Despite potential limitations, this is one of few studies conducted to determine the potential risk of paternal overweight on sperm standard parameters, sperm chromatin integrity and assisted conception outcome including fertilization, embryo quality, cleavage rate, reduce blastocyst development, implantation, and cumulative live birth rate (CLBR).MethodsA cross-sectional study of 750 infertile couples undergoing assisted reproduction technique at a single reproductive medicine center of Salma Kafeel Medical Centre Islamabad. Sperm from men undergoing ART were analyzed for chromatin integrity using sperm chromatin dispersion assay (SCD), Chromomycin A3 staining (CMA3), and toluidine blue (TB) staining, while other semen parameters were assessed on same day includes; standard semen parameters, reactive oxygen species (ROS), sperm deformity index (SDI), teratozoospermic index (TZI), and hypo-osmatic swelling test (HOST). Paternal body mass index (BMI) < 24.5–20 kg/m2 served as the reference group, while the male patients with BMI > 24.5-30 kg/m2 were considered to be overweight.ResultsIn the analysis of the percentage of spermatozoa with chromatin maturity (CMA3) and chromatin integrity (TB) was reduced significantly in overweight men (p < 0.01) compared with a reference group. Increase in paternal BMI correlate with the increase in sperm chromatin damage (SCD r = 0.282, TB r = 0.144, p < 0.05), immaturity (CMA3, r = 0.79, p < 0.05) and oxidative stress (ROS) (r = 0.282, p < 0.001). Peri-fertilization effects were increased in oocytes fertilization in couples with overweight men (FR = 67%) compared with normal-weight men (FR = 74.8%), similarly, after univariant regression paternal weight remain predictor of sperm chromatin maturity, successful fertilization and CLBR. In the embryo, developmental stage number of the embryo in cleavage was higher in normal weight men, while day 3 (D3) embryos, percent good quality embryo D3, and blastocyst formation rate were compared able between the groups. The paternal overweight group had significant (p < 0.001) increased neonatal birth weight (2952.14 ± 53.64gm; within normal range) when compared with the reference group (2577.24 ± 30.94gm) following assisted reproductive technology (ART). CLBR was higher (p < 0.05) in normal weight men compared to couples with overweight male partners. CLBR per embryo transfer and per 2PN was a statistically significant (p < 0.05) difference between the two groups. An inverse association was observed in the linear regression model between paternal BMI with fertilization rate and CLBR.ConclusionThe present study demonstrated the impact of paternal overweight on male reproductive health, as these patients had a higher percentage of immature sperm (CMA3) with impaired chromatin integrity (SCD, TB) in their semen and had decreased fertilization rate, CLBR following assisted reproductive treatments. The present study supports that paternal overweight should be regarded as one of the predictors for fertilization, CLBR and useful for counseling, to consider body mass index not only in women but also for men, in those couples opting for ART treatment, and warrant a poor reproductive outcome in overweight men.

Modelling of anaerobic digester for the conversion of organic waste into hydrogen & methane

Multistage Anaerobic digestion (AD) nowadays plays a beneficial role compared to single-stage AD to achieve a high rate of waste conversion with higher energy recovery. Hence, obtaining a stable operation with optimal operating conditions is of paramount importance in the modeling study. In the current study, modeling of two stages continuous stirred anaerobic digesters was considered using mass balances over the substrates (organic waste) and biomass in the respective reactor. All the simulation work was performed using MATLAB, from which steady-state concentration values were taken for the calculation of Hydrogen flow rate (QH2) and Methane flow rate (QCH4). The operating volumes of both the reactors were computed by looking at the effect on the production rates (QH2 &QCH4) as a function of dilution rate of first (D1) and second (D2) reactors for constant initial substrate concentration (S0). At last, the effect of Initial substrate concentration on both QH2 &QCH4 was studied by considering different S0 values as 4 g/L, 8 g/L, and 12 g/L. From our modeling result, it was found out that for Maximum Hydrogen production, operating D1 (in day-1) was 0.8, 0.9 & 1 and the Methane production rate was Maximum at operating D2 (inday-1) of 0.25, for S0 values of 4 g/L, 8 g/L, and 12 g/L respectively.

Phosphatidylcholine and Phosphatidylserine Uniquely Modify the Secondary Structure of α-Synuclein Oligomers Formed in Their Presence at the Early Stages of Protein Aggregation.

Abrupt aggregation of α-synuclein (α-Syn) leads to a formation of highly toxic protein oligomers. These aggregates are the underlying molecular cause of an onset of the irreversible degeneration of dopaminergic neurons in midbrain, hypothalamus, and thalamus, a pathology known as Parkinson's disease. The transient nature of oligomers, as well as their structural and morphological heterogeneity, limits the use of cryo-electron microscopy and solid-state NMR, classical tools of structural biology, for elucidation of their secondary structure. Despite this limitation, numerous pieces of experimental evidence suggest that phospholipids can uniquely alter the structure and toxicity of oligomers. In this study, we utilize an innovative nano-infrared imaging technique, also known as atomic force microscopy infrared (AFM-IR) spectroscopy, to examine the structure of individual α-Syn oligomers grown in the presence of phosphatidylcholine (α-Syn:PC) and phosphatidylserine (α-Syn:PS). We determined the amount of the parallel and the antiparallel β-sheets, as well as the amount the α-helix and the unordered protein, in the secondary structure of α-Syn:PC and α-Syn:PS formed at day 2 (D2), 8 (D8), and 15 (D15) after initiation of protein aggregation. We found a gradual decrease in the amount of the parallel β-sheet in both α-Syn:PC and α-Syn:PS from D2 to D15 together with an increase in the α-helix and the unordered protein secondary structure. We infer that this is due to the presence of lipids in the structure of oligomers that prevent an expansion of the parallel β-sheet upon interaction of the oligomers with monomeric α-Syn.

Chymotrypsin attenuates adjuvant-induced arthritis by downregulating TLR4, NF-κB, MMP-1, TNF-α, IL-1β, and IL-6 expression in Sprague–Dawley rats

Objective Rheumatoid arthritis (RA) is mainly characterized by synovial hyperplasia, angiogenesis, inflammatory cells infiltration. Chymotrypsin is a proteolytic enzyme with anti-inflammatory effects. The current project was intended to test the efficacy and mechanism of chymotrypsin in adjuvant-induced arthritis (AIA) rats to provide an experimental basis for the clinical application of chymotrypsin. Methods Sprague–Dawley rats were injected with complete Freund’s adjuvant (CFA) in the hind left paw pad to establish an AIA model. Forty rats were randomly divided into five groups (n = 8): blank; CFA model (model); low-dose chymotrypsin (CLD), 0.53 mg/kg; high-dose chymotrypsin (CHD), 1.06 mg/kg; piroxicam, 10 mg/kg. The treatments were performed in the subplantar region of the left hind paw from Day 8 (D8) to Day 28 after adjuvant injection. The body weight, paw diameter, swelling degree of paw, and arthritic score were measured on D0, D7, D14, D21, and D28. All animals were sacrificed on D29. Subsequently, the synovial tissue of the ankle joint of the rats was stained with HE to generate pathological sections for observation of the pathological changes of synovial tissue from the ankle joint. The protein levels of MMP-1, TNF-α, IL-1β, and IL-6 in the rats’ serum were determined by ELISA. Western blotting was used to detect the protein expression of TLR4 and NF-κB in the rat ankle tissue. The mRNA expression of TLR4, NF-κB, IL-1β, IL-6, and TNF-α in synovial tissue of the ankle joint was detected by RT-qPCR. Results The body weight of the rats in each group showed an increasing trend, and there was no significant difference in weight between the groups. CHD and piroxicam suppressed paw swelling and arthritic scores and decreased synovial hyperplasia, inflammatory cell infiltration, pannus formation, and bone destruction. Furthermore, the overproduction of MMP-1, TNF-α, IL-1β, and IL-6 in serum was remarkably attenuated in the chymotrypsin- and piroxicam-treated rats. The protein levels of TLR4 and NF-κB in the synovial tissue of the chymotrypsin group and the piroxicam group were significantly lower than those in the model group. Likewise, the rats treated with chymotrypsin and piroxicam had a substantial decline in the mRNA expression of TLR4, NF-κB, TNF-α, IL-1β, and IL-6 in synovial tissue. Conclusions Chymotrypsin alleviates the joint damage of AIA rats, probably by reducing the expression of MMP-1, TNF-α, IL-1β, and IL-6 through TLR4/NF-κB signaling pathway.