Sterile Environment Research Articles

Natural intraepithelial lymphocyte populations rise during necrotic enteritis in chickens.

Intraepithelial lymphocytes (IEL) reside in the epithelium at the interface between the contents of the intestinal lumen and the sterile environment of the lamina propria. Because of this strategic location, IEL play a crucial role in various immunological processes, ranging from pathogen control to tissue stability. In mice and humans, IEL exhibit high diversity, categorized into induced IEL (conventional CD4 and CD8αβ T cells) and natural IEL (TCRαβCD8αα, TCRγδ, and TCRneg IEL). In chickens, however, the subpopulations of IEL and their functions in enteric diseases remain unclear. Thus, we conducted this study to investigate the role of IEL populations during necrotic enteritis (NE) in chickens. At 14 days of age, sixty-three Specific-pathogen-free (SPF) birds were randomly assigned to three treatments: Control (sham challenge), Eimeria maxima challenge (EM), and Eimeria maxima + Clostridium Perfringens (C. Perfringens) co-challenge (EM/CP). The EM and EM/CP birds were infected with Eimeria maxima at day 14 of age, and EM/CP birds were additionally orally inoculated with C. perfringens at days 18 and 19 of age. Birds were weighed at days 18, 20, and 26 of age to assess body weight gain (BWG). At 20 days of age (1 day-post C. perfringens infection; dpi), and 26 days of age (7 dpi), 7 birds per treatment were euthanized, and jejunum was harvested for gross lesion scores, IEL isolation, and gene expression. The EM/CP birds exhibited subclinical NE disease, lower BWG and shorter colon length. The Most changes in the IEL populations were observed at 1 dpi. The EM/CP group showed substantial increases in the total number of natural IEL subsets, including TCRαβ+CD4-CD8-, TCRαβ+CD8αα+, TCRγδ+, TCRneg and innate CD8α (iCD8α) cells by at least two-fold. However, by 7 dpi, only the number of TCRαβ+CD4-CD8- and TCRαβ+CD8αα+ IEL maintained their increase in the EM/CP group. The EM/CP group had significantly higher expression of proinflammatory cytokines (IL-1β and IFN-γ) and Osteopontin (OPN) in the jejunum at 1 dpi. These findings suggest that natural IEL with innate and innate-like functions might play a critical role in the host response during subclinical NE, potentially conferring protection against C. perfringens infection.

Crosstalk between keratinocytes and neutrophils shapes skin immunity against S. aureus infection.

Staphylococcus aureus (S. aureus) infection of the skin leads to a rapid initial innate immune response with keratinocytes in the epidermis as the initial sensors. Polymorphonuclear neutrophils (PMNs) are the first innate immune cells to infiltrate infection sites where they provide an effective first-line of defense. Previous work of our group showed that in inflamed skin a crosstalk between PMNs and keratinocytes results in enhanced S. aureus skin colonization. In this work, we used an in vitro co-culture model to studied the crosstalk between primary human keratinocytes (PHKs) and PMNs in a sterile environment and upon S. aureus infection. We investigated the influence of PHKs on PMN activation by analyzing PMN lifespan, expression of degranulation markers and induction of proinflammatory cytokines. Furthermore, we analyzed the influence of PMNs on the inflammatory response of PHKs. Finally, we investigated the influence of the skin microbiome on PMN-mediated skin inflammation. We show that co-culture of PMNs with PHKs induces activation and degranulation of PMNs and significantly enhances their lifespan compared to PMN cultivation alone by an IL-8 mediated mechanism and, furthermore, primes PMNs for enhanced activity after S. aureus infection. The prolonged incubation with PMNs also induces inflammatory responses in PHKs which are further exacerbated in the presence of S. aureus and induces further PMN recruitment thus fueling skin inflammation. Interestingly, infection of PHKs with the skin commensal S. epidermidis reduces the inflammatory effects of PMNs in the skin and exhibits an anti-inflammatory effect. Our data indicate that skin infiltrating PMNs and PHKs influence each other in such a way to enhance skin inflammation and that commensal bacteria are able to reduce the inflammatory effect.

How to Evaluate If Microorganisms Isolated From Sterile Drug Production Environments Monitoring Are Undesirable

This study addresses the identification of undesirable microorganisms (MOs) recovered during the environmental monitoring in manufacture of sterile medicinal products. We developed a methodology evaluation based on a decision tree; then, such approach was applied to hypothetical scenarios of uncommon MOs isolation in sterile drugs production settings. The scenarios were formulated on the basis of our field experience, in terms of both MOs selection and types of sampling site. The MOs were chosen in order to include emerging pathogens and MOs responsible for drug recall, and several sampling sites were considered for their detection (air, surfaces, and personnel). The classification of the unusual MOs revealed that most of them were undesirable, because they represented the loss of environmental control or a potential impact on the quality of the product. In some cases, the uncommon MOs were not considered as undesirable. Therefore, our results demonstrated the importance of a methodology, also in terms of recovery rate of unusual MOs and of the threshold probability for the unacceptability (e.g., 1% or 5%). The proposed methodology allowed an easy and documented evaluation for the undesirable MOs isolated from the environment of the analyzed settings for sterile drugs production.

Bacterially enhanced plant-growing media for controlled environment agriculture.

Microbe-plant interactions in the root zone not only shape crop performance in soil but also in hydroponic cultivation systems. The biological and physicochemical properties of the plant-growing medium determine the root-associated microbial community and influence bacterial inoculation effectiveness, which affects plant growth. This study investigated the combined impact of plant-growing media composition and bacterial community inoculation on the root-associated bacterial community of hydroponically grown lettuce (Lactuca sativa L.). Ten plant-growing media were composed of varying raw materials, including black peat, white peat, coir pith, wood fibre, composted bark, green waste compost, perlite and sand. In addition, five different bacterial community inocula (BCI S1-5) were collected from the roots of lettuce obtained at different farms. After inoculation and cultivation inside a vertical farm, lettuce root-associated bacterial community structures, diversity and compositions were determined by evaluating 16S rRNA gene sequences. The study revealed distinct bacterial community structures among experimental replicates, highlighting the influence of raw material variations on root-associated bacterial communities, even at the batch level. However, bacterial community inoculation allowed modulation of the root-associated bacterial communities independently from the plant-growing medium composition. Bacterial diversity was identified as a key determinant of plant growth performance with green waste compost introducing Bacilli and Actinobacteria, and bacterial community inoculum S3 introducing Pseudomonas, which positively correlated with plant growth. These findings challenge the prevailing notion of hydroponic cultivation systems as sterile environments and highlight the significance of proper plant-growing media raw material selection and bacterial community inoculation in shaping root-associated microbiomes that provide stability through microbial diversity. This study supports the concept of creating bacterially enhanced plant-growing media to promote plant growth in controlled environment agriculture.

Early-Life Fecal Transplantation from High Muscle Yield Rainbow Trout to Low Muscle Yield Recipients Accelerates Somatic Growth through Respiratory and Mitochondrial Efficiency Modulation.

Previous studies conducted in our lab revealed microbial assemblages to vary significantly between high (ARS-FY-H) and low fillet yield (ARS-FY-L) genetic lines in adult rainbow trout. We hypothesized that a high ARS-FY-H donor microbiome can accelerate somatic growth in microbiome-depleted rainbow trout larvae of the ARS-FY-L line. Germ-depleted larvae of low ARS-FY-L line trout reared in sterile environments were exposed to high- or low-fillet yield-derived microbiomes starting at first feeding for 27 weeks. Despite weight-normalized diets, somatic mass was significantly increased in larvae receiving high fillet yield microbiome cocktails at 27 weeks post-hatch. RNA-seq from fish tails reveals enrichment in NADH dehydrogenase activity, oxygen carrier, hemoglobin complex, gas transport, and respiratory pathways in high fillet yield recolonized larvae. Transcriptome interrogation suggests a relationship between electron transport chain inputs and body weight assimilation, mediated by the gut microbiome. These findings suggest that microbiome payload originating from high fillet yield adult donors primarily accelerates juvenile somatic mass assimilation through respiratory and mitochondrial input modulation. Further microbiome studies are warranted to assess how increasing beneficial microbial taxa could be a basis for formulating appropriate pre-, pro-, or post-biotics in the form of feed additives and lead to fecal transplantation protocols for accelerated feed conversion and fillet yield in aquaculture.

The Interplay between Mycobacterium tuberculosis and Human Microbiome.

Tuberculosis (TB), a respiratory disease caused by Mycobacterium tuberculosis (Mtb), is a significant cause of mortality worldwide. The lung, a breeding ground for Mtb, was once thought to be a sterile environment, but has now been found to host its own profile of microbes. These microbes are critical in the development of the host immune system and can produce metabolites that aid in host defense against various pathogens. Mtb infection as well as antibiotics can shift the microbial profile, causing dysbiosis and dampening the host immune response. Additionally, increasing cases of drug resistant TB have impacted the success rates of the traditional therapies of isoniazid, rifampin, pyrazinamide, and ethambutol. Recent years have produced tremendous research into the human microbiome and its role in contributing to or attenuating disease processes. Potential treatments aimed at altering the gut-lung bacterial axis may offer promising results against drug resistant TB and help mitigate the effects of TB.

Sustainability in the Breast Operating Theatre: Promoting a Multidisciplinary Sustainability strategy across multiple centres

Operating theatres in hospitals are critical areas that generate a significant amount of waste, including single-use items such as disposable hats which has a significant effect on our NHS carbon footprint. The use of appropriate surgical attire, including hats, plays a vital role in maintaining hygiene and promoting a sterile environment within operating theatres. The introduction of the RCS surgical green checklist has highlighted several ways to improve sustainability in a theatre setting.

Skin Care Experience of a Case of Multiple Organ Failure Caused by High-Voltage Electrical Injury

Background: High-voltage electrical burns are caused by the direct transmission of electric current through the body and the conversion of electric energy into heat energy, which directly heats the tissues and leads to organ function damage. Especially after high-voltage electric burns (more than 1000W), it can not only cause coagulation necrosis of the skin and even carbonization, but also cause serious deep tissue damage .The degree of tissue damage depends on the type of current, the magnitude of current, the frequency, the voltage, the resistance of the tissue, the skin humidity, the duration, the contact area, and other factors. Low voltage and low frequency electric shock mainly affect the heart and respiration, which can lead to insignificant soft tissue damage. High voltage electric shock mainly causes serious soft tissue damage, which can be accompanied by changes in the circulatory system and respiratory system.[1] Subject and Method: We collected the data of a case of multiple organ failure caused by high-voltage electricity that was successfully treated in July 2023.In the course of this patient's successful treatment, The main nursing measures were analyzed: The intake and output volume were actively controlled. Albumin supplementation, Debridement and dressing change were performed daily, Covered with nano-silver ion dressing, Try to ensure a sterile environment during operation, Maintain ambient temperature and humidity. Results: Twenty days later,the skin damage caused by electric burns was reduced from 30% to 1%. Conclusion: These are very important links: The intake and output volume were actively controlled, The balance of intake and output was maintained for 24 hours, and lactic acid was less than 2mmol/L. Albumin supplementation Controlled above 30g/L, Debridement and dressing change were performed daily, Covered with nano-silver ion dressing, Try to ensure a sterile environment during operation, Maintain ambient temperature 28~30℃ and humidity 40%~50%.

Impact of the practice of touch-massage® by a nurse on the anxiety of patients with hematological disorders hospitalized in a sterile environment, a randomized, controlled study

ContextIn addition to curative care, supportive care is beneficial in managing the anxiety symptoms common in patients in sterile hematology unit. We hypothesize that personal massage can help the patient, particularly in this isolated setting where physical contact is extremely limited. The main objective of this study was to show that anxiety could be reduced after a touch-massage® performed by a nurse trained in this therapy.MethodsA single-center, randomized, unblinded controlled study in the sterile hematology unit of a French university hospital, validated by an ethics committee. The patients, aged between 18 and 65 years old, and suffering from a serious and progressive hematological pathology, were hospitalized in sterile hematology unit for a minimum of three weeks, patients were randomized into either a group receiving 15-minute touch-massage® sessions or a control group receiving an equivalent amount of quiet time once a week for three weeks. In the treated group, anxiety was assessed before and after each touch-massage® session, using the State-Trait Anxiety Inventory questionnaire with subscale state (STAI-State). In the control group, anxiety was assessed before and after a 15-minute quiet period. For each patient, the difference in the STAI-State score before and after each session (or period) was calculated, the primary endpoint was based on the average of these three differences. Each patient completed the Rosenberg Self-Esteem Questionnaire before the first session and after the last session.ResultsSixty-two patients were randomized. Touch-massage® significantly decreased patient anxiety: a mean decrease in STAI-State scale score of 10.6 [7.65–13.54] was obtained for the massage group (p ≤ 0.001) compared with the control group. The improvement in self-esteem score was not significant.ConclusionThis study provides convincing evidence for integrating touch-massage® in the treatment of patients in sterile hematology unit.Trial registrationNCT02343965.

Nekomplikovane infekcije mokraćnih puteva kod starih osoba

A healthy urinary tract is not a sterile environment but is inhabited by diverse microorganisms that change according to environmental factors and the patient's condition. Elderly patients are predisposed to urinary tract infections and asymptomatic bacteriuria, and distinguishing between these two conditions is challenging. They are specific in several issues: the pathogenesis of infection and causative agents, diagnostic procedures, applied therapy, and preventive measures. Proper interpretation of symptoms, sampling of materials for analysis, and interpretation of results are of great importance. Correct procedures allow sparing patients from unnecessary treatment and complications of antibiotic therapy, as well as reducing bacterial resistance.

Patient-Derived Xenograft Models for Leukemias.

Patient-derived xenograft (PDX) modeling is a valuable tool for the study of leukemia pathogenesis, progression, and therapy response. Engraftment of human leukemia cells occurs following injection into the tail vein (or retro-orbital vein) of preconditioned immunocompromised mice. Injected mice are maintained in a sterile and supportive housing environment until leukemia engraftment is observed, at which time studies such as drug treatments or leukemia sampling can occur. Here, we outline a method for generating PDXs from Acute Myeloid Leukemia (AML) patient samples using tail vein injection; however it can also be readily applied to T- and B- Acute Lymphoblastic Leukemia (ALL) samples.

Improvised Femoral Nerve Block Model Using Everyday Items: An Affordable Alternative for Medical Training

Background : Nerve blocks have emerged as a revolutionary approach for managing pain and reducing opioid dependence among patients. Performing nerve blocks under ultrasound guidance in the emergency department (ED) is considered an effective and minimally invasive procedure that can be performed by adequately trained ED physicians. However, the cost associated with commercially available nerve block models poses a significant challenge to widespread adoption and training. To address this issue, our research team developed an improvised femoral nerve block model using readily available everyday items. Methods : The prototype femoral nerve block model was constructed using chicken breasts as a simulated anatomical structure, balloons and spaghetti as tactile representations of vessels and nerves, gel as an acoustic interface, and cling wrap for creating a sterile environment. Results : To perform ultrasound-guided nerve blocks, it is necessary to see the fascial plane division clearly while injecting the anesthetic. This clarity was accomplished by using two layers of chicken breasts, each wrapped individually in cling wrap. Additionally, it is crucial to visualize and differentiate the nerve, artery, and vein. This was achieved by filling the balloon with different substances that served as distinct markers for each vessel. Preliminary results demonstrated that the improvised femoral nerve block model effectively simulated the anatomical and tactile characteristics necessary for nerve block training. Conclusion : In conclusion, our low-fidelity femoral nerve block model offers an affordable and accessible option for training healthcare professionals in ultrasound-guided nerve blocks. By utilizing everyday items, we have demonstrated that cost-effective alternatives can be developed to address the financial constraints associated with conventional training models. Further research is warranted to refine and validate this improvised model and explore its potential applications in various medical specialties.

In vitro cultivation of Vismia japurensis: Isolation of the new anthrone 1,8,10-trihydroxy-3,10-dimethyl-9(10H)-anthracenone.

Vismia japurensis Reichardt is a plant of ecological and chemical importance from which a variety of bioactive substances have been isolated. The current study aimed to establish in vitro cultures of this species as a source of secondary metabolites. Appropriate decontamination treatments and germination tests were performed and, after in vitro culture establishment, the propagated plants were multiplied in a sterile environment to increase the biomass of available experimental material. Seeds showed low contamination and a high germination percentage on Woody Plant Medium (WPM) supplemented with gibberellic acid (both at concentrations of 5 and 10 mg/L). V. japurensis nodal segments rapidly regenerated when first grown in WPM and then transplanted to Murashige and Skoog medium (MS). After 60 days in MS medium, the propagated plants were removed, lyophilized, and extracted with hexane and methanol. The hexane extract was fractionated via open column chromatography, and the substance isolated was purified by high performance liquid chromatography. Structural determination of the isolated substance was carried out using one and two-dimensional nuclear magnetic resonance and mass spectrometry. The isolated substance was identified as 1,8,10-trihydroxy-3,10-dimethyl-9(10H)-anthracenone, which, based on the conducted literature search, is reported for the first time.

Сравнительный анализ микробиоты ткани яичка и уретры у пациентов с азооспермией методом секвенирования нового поколения (NGS)

Aim. To determine the role of taxonomic bacterial diversity of the urethral and testicular microbiota in the development of male infertility using high-throughput next-generation sequencing (NGS). Material and methods. In the period between 2018 and 2022 we examined 53 patients. Three study groups were formed: Infertile patients with nonobstructive azoospermia and concomitant varicocele (n = 13) – group 1; Infertile patients with obstructive azoospermia (n = 29) – group 2; Fertile patients with confirmed paternity and testicular pathology requiring histological verification to exclude testicular oncopathology (n = 11) – group 3. Each patient underwent TESE with testicular both and urethral tissue sampling in order to compare the bacterial landscape and control the purity of the method. The obtained material was sequenced using a high-throughput method (NGS). Results. According to our findings, the testicular tissue of obstructive azoospermia patients had significantly depleted bacterial diversity in comparison to nonobstructive azoospermia and concomitant varicocele ones. Also, the fertile group turned out to be the most diverse in its taxonomic community. These results may suggest the bacterial microbiome’s influence on men’s reproductive health. Discussion. Our urogenital bacterial diversity analysis showed that human testicular tissue is not a microbiologically sterile environment and also presented new data associated with testicular tissue and its possible relations with male infertility. Conclusion. A focus on the qualitative and quantitative composition of the testicular microbial community may form the new basis in the diagnosis and treatment of male infertility associated with various types of azoospermia

Case series on two-stage disinfection in regenerative endodontics on permanent mandibular molar with blunderbuss canal

Background: Revascularization is a promising approach as it promotes root end closure forming healthy, thick root dentin at the apex which provides clinical and radiographic success due to the elimination of bacteria and disinfection of the canal which gives a 100% sterile environment to receive SCAP. Aim: To examine the effect of a regenerative endodontic procedure on immature necrotic teeth with apical periodontitis Method: An 8-year-old medically fit boy was referred to the department with a lingering type of pain in the immature permanent mandibular first right molar. Bleeding was induced in the root canals at the third appointment. The blood clots (scaffolds) were covered with MTA, and the tooth was restored with a composite filling. Recall sessions were scheduled after one month and 3 months, when the tooth appeared asymptomatic clinically and radiographically, with considerable periapical healing. Conclusion: An immature multirooted tooth with necrotic pulps and periapical radiolucency was managed with evident clinical and radiographic success, wherein non-setting calcium hydroxide paste and triple antibiotic paste were used sequentially to guarantee complete canal disinfection.

Neonatal CD4+ T cells have a characteristic transcriptome and epigenome and respond to TCR stimulation with proliferation and yet a limited immune response.

The adaptive immune response is coordinated by CD4+ T cells, which determine the type and strength of the immune response and the effector cells involved. It has been reported that CD4+ T cells are less responsive in neonates, leading to low activation of the cellular response and poor antibody production by B cells. This low response is essential for the tolerant window that favors birth transition from the sterile environment in the womb to the outside world but leaves neonates vulnerable to infection, which is still an important health issue. Neonates have a high morbidity and mortality rate due to infections, and the molecular reasons are still understudied. We asked whether the neonatal naive CD4+ T cells have a genomic program that predisposes them to a low response. Therefore, we evaluated the transcriptome and epigenome of human neonatal and adult naive CD4+ T cells. Our results point to a gene expression profile forming a distinct regulatory network in neonatal cells, which favors proliferation and a low T-cell response. Such expression profile is supported by a characteristic epigenetic landscape of neonatal CD4+ T cells, which correlates with the characteristic transcriptome of the neonatal cells. These results were confirmed by experiments showing a low response to activation signals, higher proliferation, and lower expression of cytokines of neonatal CD4+ T cells as compared to adult cells. Understanding this network could lead to novel vaccine formulations and better deal with life-threatening diseases during this highly vulnerable period of our lives.

Protective Airflow including ‘First Air’ in GMP Aseptic manufacturing

Good Manufacturing Practices (GMP) related to sterile product aseptic manufacturing environments rely on protective airflow mechanisms to ensure the control over airborne contamination. Alongside the protective airflow, pressure differentials that drive airflow cascade ensure GMP compliant contamination control of airborne contamination that may include Microbial carrying particles (MCPs). Airflow visualisation qualification by smoke studies has been clarified as a GMP requirement in EU & PICS Annex 1. The scope of Annex 1 applies to manufacture of sterile medicinal products together with bioburden control processes where bioburden in intermediates, substances, APIs and non-sterile products can also impact patient safety so a wide scope of protective airflow applications is applied. As a GMP requirement (included in 2008 version of Annex 1): It should be demonstrated that air-flow patterns do not present a contamination risk, e.g. care should be taken to ensure that air flows do not distribute particles from a particle generating person, operation or machine to a zone of higher product risk.' Within Annex 1: 2023, the concept of First Air has emerged as a critical element. First Air is defined as “filtered air that has not been interrupted prior to contacting exposed product and product contact surfaces with the potential to add contamination to the air prior to reaching the critical zone.” The requirement for extensive smoke studies and the integration of First Air protection as a mandated aspect in GMP regulations has brought to the fore an imperative need for a deeper comprehension of airflow dynamics and their implications on sterile product Aseptic manufacturing. This shift beckons us to explore the nuances of protective airflow, particularly in the context of Computational Fluid Dynamics (CFD) analyses, to discern the efficacy of airflow patterns in different scenarios. Stakeholders involved in Annex 1 implementation have been impacted by introduction of the First Air protection requirement, considering design and in qualification via smoke study airflow visualisation. Such requirements must follow QRM principles where process understanding and knowledge of contamination hazards are essential to mitigate risks in compromise of product quality and potential harm to patients. Risk mitigations must take a Quality by Design (QbD) approach as monitoring alone does not provide assurance of product sterility or in meeting defined bioburden limits.

Managing Addiction and Overdose Deaths: The Debate Over America's Safe Injection Spaces.

In the decade between 2010 and 2020, the number of people killed by opioid overdoses more than tripled, reaching 68,630. Among drug users in their second and third decades of life, hepatitis B and C are also on the rise. New York City's two new supervised drug injection sites (SDISs) reversed 114 overdoses within two months, and 585 people injecting drugs visited the facility 4,974 times. By providing medical professionals and a sterile and safe environment, supervised injection sites reduce overdose risk and save lives. This suggests that SDISs could be an important adjunct to currently failing strategies to combat the nation's raging opioid crisis, particularly in the aftermath of the COVID-19 pandemic. Here, we examine the perception and impact of SDISs as well as efforts by the United States Congress (H.R. 7029 and H.R. 6159) toward their prevention. We look into the perspectives of major stakeholders, such as residents, business owners, drug users, legislators, taxpayers, and the general public, and investigate the short- and long-term consequences of SDISs based on crime statistics and published data on opioid use, overdose deaths, and blood-borne disease transmission rates.

Synergistic Growth Inhibition of NPM1 Mutant AML PDX By Combined Therapy with BCL-2 Inhibitor Venetoclax (ABT-199) and Menin Inhibitor DS-1594b In Vivo

Introduction: Mixed-lineage-leukemia (MLL) translocations are prevalent in hematologic malignancies, resulting in aggressive leukemias resistant to standard therapies and low 5-year survival rates. These translocations lead to the production of MLL fusion proteins that promote leukemia development by enhancing proliferation, inhibiting hematopoietic differentiation, and driving leukemogenesis. Acute myeloid leukemia (AML), an aggressive blood cancer with a grim prognosis, particularly in elderly patients, frequently involves mutations in the nucleophosmin (NPM1) gene. Importantly, the interaction between Menin, MLL, and MLL fusion proteins play a crucial role in the pathogenesis of AML, both with MLL rearrangements (MLLr) and in leukemias carrying NPM1 mutations. Inhibition of the Menin-MLL interaction using novel Menin inhibitors has demonstrated efficacy in pre-clinical and clinical studies for both AML subsets. In this study, we investigate the effectiveness of the novel Menin-MLL inhibitor DS-1594b, developed by Daiichi Sankyo Co. Ltd., in combination with the BCL-2 antagonist venetoclax (ABT-199), in a patient-derived xenograft (PDX) model of NPM1-mutated acute leukemia. We have previously reported synergistic lethality and differentiation-inducing effects of this drug combination in both AML cell lines and primary leukemia samples in vitro (Ciaurro et al., Blood 2022). Our findings shed light on the potential of this therapeutic combination for the targeted treatment of AML with NPM1 mutations. Methods: NSG mice (female, 8-10 weeks old) were purchased from Jackson Laboratory. Mice were inoculated via the tail vein with NPM1m PDX/luc/GFP (3×10 6 cells). Once leukemia engraftment was confirmed by bioluminescent imaging mice were randomized in 4 groups: vehicle, ABT199, DS-1594b alone, and combination. After 12 days drugs were administered as follows: DS1594b at a dosage of 50 mg/kg by oral gavage for 4 weeks; ABT-199 at a dosage of 50 mg/kg by oral gavage for 2 weeks and 100mg/kg for the other 2 weeks. NSG mice were euthanized by CO 2 inhalation at the endpoint of the study in accordance with IACUC protocols. BM and spleens were harvested in a sterile environment and flushed in HBSS with 1% FBS. For CyTOF data spleen cells were initially barcoded and stained with metal-conjugated antibodies according to Fluidigm's protocols and samples were analyzed by CyTOF (Helios) Mass Cytometer. Results: New in vitro data confirmed synergistic lethality on additional AML cell lines and demonstrated upregulation of PARP-C and Caspase3-C apoptosis pathways using western blot analysis. Further, additional 3 primary AML patient samples showed additive or synergistic growth inhibition when treated with combination of drugs. In the in vivo experiment with NPM1m PDX/luc/GFP AML PDX model, we found that co-treatment with DS-1594b and venetoclax for four weeks resulted in a greater reduction in AML burden compared to treatment with either agent alone or a vehicle control as shown by Bioluminescence imaging. Moreover, weight of mice in the combo group was not significantly affected, while mice lost weight in vehicle and in single agent cohort due to rapid disease progression. The survival curve clearly demonstrated the benefits of the combination treatment, with the DS-1594b group having modest anti-tumor efficacy, and the combination group showing the greatest efficacy (Fig. 1A). To study the effects on cell differentiation, we used CyTOF technique, which allowed us to analyze a complete panel of 30 markers. We found two populations of cells, which we named populations (pop) 3 and pop 4, that were enriched in the group treated with the combination of drugs compared to the control group (Fig 1B). These populations of cells had higher levels of expression of CD33 and CD38, which are well-known markers of cellular differentiation. Conclusions: In this study, we present the in vivo effectiveness of DS-1594b in combination with venetoclax in a PDX model of NPM1-mutated AML. Moreover, the combination treatment exhibits differentiation-inducing effects, which contribute to its therapeutic effectiveness, and was safe. Overall, our findings strongly indicate that the combination of DS-1594b and venetoclax exhibits promising anti-leukemic activity in vivo and holds potential as a therapeutic strategy for AML patients with mtNPM1 mutations.

Identification and validation of major-effect quantitative trait locus QMS-5B associated with male sterility in photo-thermo-sensitive genic male sterile wheat.

QMS-5B, a major QTL for photo-thermo-sensitive genic male sterility in wheat, was fine mapped in a 2.15Mb region harboring a serine/threonine protein kinase gene TraesCS5B03G0887500, which was the most likely candidate gene. Genic male sterility is an essential trait in the utilization of heterosis and hybrid seed production for wheat. Currently, genic male sterile genes have been reported in wheat mutants, but the sterile genes controlling photo-thermo-sensitive genic male sterility in wheat have not been studied systematically. Here, 235 doubled haploid lines derived from a cross between photo-thermo-sensitive genic male sterile line BS462 and its restorer line CP279 were used to map male sterile gene by GenoBaits® Wheat 100K Panel, bulked segregant exome sequencing (BSE-Seq) and wheat 660K array. As a result, the major stable QTL on chromosome 5B, QMS-5B, was identified in all four environments, accounting for 7.3-36.4% of the phenotypic variances. Ulteriorly, QMS-5B was delimited to an approximate 2.15Mb physical interval between KASP-5B5 and KASP-5B6 using kompetitive allele-specific PCR (KASP) markers. Within the interval, twenty-nine high-confidence genes were predicted according to Chinese Spring RefSeq v2.1. TraesCS5B03G0887500, encoding a serine/threonine protein kinase, was identified as the most likely candidate gene for QMS-5B based on weighted gene co-expression network analysis. Expression analysis confirmed that TraesCS5B03G0887500 was significantly differentially expressed in anthers of BS462 and CP279 at different stages under fertile and sterile environments. In addition, flanking KASP marker KASP-5B6 can effectively genotype male sterile lines and restorer lines, and can be used for molecular marker-assisted selection. This study provides insights into for exploring the mechanism of photo-thermo-sensitive genic male sterility in wheat.