T helper (Th) cell profiles and cytokines/chemokines in characterization, treatment, and monitoring of autoimmune diseases.

Autoimmune diseases (AD) consist of a spectrum of disease entities whose etiologies are very complex and still not well understood. Every individual has the potential for developing AD under appropriate conditions because the body contains lymphocytes that are potentially reactive with self-antigens. The aims of this study are to (1) explore the flow cytometry method to identify the frequency of various circulating CD4+ T helper (Th) cell-subsets, including Th1, Th2, Th9, Th17, Th17.1, and Th22; (2) In parallel, to examine multiplex ELISA method for pathogenic inflammatory cytokines/chemokines, and (3) To assess the correlation of expression of T cell-subsets with serum cytokines/chemokines and understand its clinical importance with available AD treatments. We analyzed Th17, Th17.1, Th22, Th2, Th1, and Th9 Th cell populations and compared the concentrations of 67 cytokines/chemokines in healthy as well as AD-diagnosed patients. We observed that patients with autoimmune markers had significantly elevated percentages of naïve (Th17, Th22, and Th9) as well as memory (Th17 and Th22) Th cell-subsets, along with increased concentrations of cytokines/chemokines (Eotaxin, TNFß, and FABP4). The percentage of Th cell-subsets correlated positively or negatively with the production of cytokines/chemokines of patients diagnosed with AD. Our study demonstrates that the naïve and memory Th cell-subsets with positive correlations to cytokines/chemokines show new diagnostic markers to predict the patients' outcome, while the negative correlation of cytokines/chemokines shows the response to autoimmune therapies. Our findings of Th cell-subsets by flow cytometry and cytokines/chemokines by multiplex ELISA suggest that CCR6+ Th cell-subsets (Th17, Th17.1, Th22, and Th9) contribute to our understanding of the pathogenesis of AD and identify the new onset of AD from the autoimmune spectrum. Our findings highlight the importance of CCR6+ as a possible marker in the characterization, treatment, and monitoring of AD.

Purified recombinant lentiviral Vpx proteins maintain their SAMHD1 degradation efficiency in resting CD4+ T cells.

Different protein purification methods exist. Yet, they need to be adapted for specific downstream applications to maintain functional integrity of the recombinant proteins. This study established a purification protocol for lentiviral Vpx (viral protein X) and test its ability to degrade sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1) ex vivo in resting CD4+ T cells. For this purpose, we cloned a novel eukaryotic expression plasmid for Vpx including C-terminal 10x His- and HA-tags and confirmed that those tags did not alter the ability to degrade SAMHD1. We optimized purification conditions for Vpx produced in HEK293T cells with CHAPS as detergent and Co-NTA resins yielding the highest solubility and protein amounts. Size exclusion chromatography (SEC) further enhanced the purity of recombinant Vpx proteins. Importantly, nucleofection of resting CD4+ T cells demonstrated that purified recombinant Vpx protein efficiently degraded SAMHD1 in a proteasome-dependent manner. In conclusion, this protocol is suitable for functional downstream applications of recombinant Vpx and might be transferrable to other recombinant proteins with similar functions/properties as lentiviral Vpx.

Highly efficacious and safe neutralizing DNA aptamer of SARS-CoV-2 as an emerging therapy for COVID-19 disease.

BACKGROUND: The paucity of SARS-CoV-2-specific virulence factors has greatly hampered the therapeutic management of patients with COVID-19 disease. Although available vaccines and approved therapies have shown tremendous benefits, the continuous emergence of new variants of SARS-CoV-2 and side effects of existing treatments continue to challenge therapy, necessitating the development of a novel effective therapy. We have previously shown that our developed novel single-stranded DNA aptamers not only target the trimer S protein of SARS-CoV-2, but also block the interaction between ACE2 receptors and trimer S protein of Wuhan origin, Delta, Delta plus, Alpha, Lambda, Mu, and Omicron variants of SARS-CoV-2. We herein performed in vivo experiments that administer the aptamer to the lungs by intubation as well as in vitro studies utilizing PBMCs to prove the efficacy and safety of our most effective aptamer, AYA2012004_L. METHODS: In vivo studies were conducted in transgenic mice expressing human ACE2 (K18hACE2), C57BL/6J, and Balb/cJ. Flow cytometry was used to check S-protein expressing pseudo-virus-like particles (VLP) uptake by the lung cells and test the immuogenicity of AYA2012004_L. Ames test was used to assess mutagenicity of AYA2012004_L. RT-PCR and histopathology were used to determine the biodistribution and toxicity of AYA2012004_L in vital organs of mice. RESULTS: We measured the in vivo uptake of VLPs by lung cells by detecting GFP signal using flow cytometry. AYA2012004_L specifically neutralized VLP uptake and also showed no inflammatory response in mice lungs. In addition, AYA2012004_L did not induce inflammatory response in the lungs of Th1 and Th2 mouse models as well as human PBMCs. AYA2012004_L was detectable in mice lungs and noticeable in insignificant amounts in other vital organs. Accumulation of AYA2012004_L in organs decreased over time. AYA2012004_L did not induce degenerative signs in tissues as seen by histopathology and did not cause changes in the body weight of mice. Ames test also certified that AYA2012004_L is non-mutagenic and proved it to be safe for in vivo studies. CONCLUSIONS: Our aptamer is safe, effective, and can neutralize the uptake of VLPs by lung cells when administered locally suggesting that it can be used as a potential therapeutic agent for COVID-19 management.

Haploid parthenotes express differential response to in vitro exposure of ammonia compared to normally fertilized embryos.

In the present study, we assessed whether absence of paternal genome imparts any differential response in embryos to chemical stress such as ammonia. Parthenogenesis was induced in MII stage oocytes using 10 mM SrCl2 in M16 medium. Parthenotes and normally fertilized embryos at 2 cell stage were exposed to different concentrations of ammonia and cultured till blastocyst. Exposure of ammonia to normally fertilized embryos resulted in significant decrease in the developmental potential (p < 0.0001) and blastocyst quality (p < 0.001). Whereas, in parthenotes, even though lower concentrations of ammonia did not have any effect, at 200 µM concentration the blastocyst rate was two times higher than control. The baseline apoptotic index was higher in parthenotes compared to normally fertilized embryos, which further increased after ammonium exposure (p < 0.001). Unlike in normally fertilized embryos ammonia exposure altered the mitochondrial distribution pattern and lead to increased expression of Oct4, Nanog and Na+/K+ ion exchange channel, while the cytochrome C expression was downregulated. This indicates that haploidy and/or absence of paternal factors in the embryo results in differential tolerance to stress induced by ammonia.

Supplementing zinc oxide nanoparticles to cryopreservation medium minimizes the freeze-thaw-induced damage to spermatozoa.

The sperm DNA integrity post cryopreservation of human semen samples is one of the serious concerns in human infertility treatment. In the present study, the beneficial effects of zinc oxide nanoparticles in preserving the functional ability of spermatozoa was explored. Ejaculates of normozoospermic men cryopreserved along with Zinc oxide nanoparticles (ZnONPs) exhibited non-significantly higher percentage of total and progressive motility in frozen-thawed samples compared to control. The sperm chromatin damage and malondialdehyde (MDA) level was significantly lower in ZnONPs group (P < 0.01 and P < 0.05 respectively) and the spermatozoa's ability to undergo acrosome reaction was also unaltered. Fluorescence microscopy and High resolution transmission electron microscopy analysis demonstrated that the ZnONPs do not penetrate the membrane of spermatozoa but stay around the spermatozoa. In conclusion, the presence of ZnONPs during cryopreservation appears to be beneficial to the spermatozoa as they withstand freeze-thaw process competently better than control, without any adverse effect shown.

Sperm-derived factors enhance the in vitro developmental potential of haploid parthenotes.

Parthenotes are characterized by poor in vitro developmental potential either due to the ploidy status or the absence of paternal factors. In the present study, we demonstrate the beneficial role of sperm-derived factors (SDF) on the in vitro development of mouse parthenotes. Mature (MII) oocytes collected from superovulated Swiss albino mice were activated using strontium chloride (SrCl2) in the presence or absence of various concentrations of SDF in M16 medium. The presence of SDF in activation medium did not have any significant influence on the activation rate. However, a significant increase in the developmental potential of the embryos and increased blastocyst rate (P < 0.01) was observed at 50 µg/ml concentration. Furthermore, the activated oocytes from this group exhibited early cleavage and cortical distribution of cortical granules that was similar to that of normally fertilized zygotes. Culturing 2-cell stage parthenotes in the presence of SDF significantly improved the developmental potential (P < 0.05) indicating that they also play a significant role in embryo development. In conclusion, artificial activation of oocytes with SDF can improve the developmental potential of parthenotes in vitro.

Distribution pattern of cytoplasmic organelles, spindle integrity, oxidative stress, octamer-binding transcription factor 4 (Oct4) expression and developmental potential of oocytes following multiple superovulation.

The aim of the present study was to determine the effects of repeated superovulation on oocyte quality and embryo developmental potential. Female Swiss albino mice were injected with 5IU pregnant mare's serum gonadotropin followed 48h by 10IU human chorionic gonadotropin. Mice were superovulated up to four times with a gap of 7 days between each superovulation cycle. Ovarian weight increased significantly with an increasing number of superovulation cycles. Although the first stimulation cycle resulted in a threefold increase in the number of oocytes, the number of oocytes decreased gradually after subsequent stimulations. Increased cytoplasmic fragmentation, abnormal mitochondrial distribution, aggregation of Golgi apparatus, spindle damage, increased intracellular oxidative stress and a decrease in expression of octamer-binding transcription factor 4 (Oct4) expression were observed in these oocytes. Further, embryos derived from mice subjected to multiple stimulation cycles exhibited a low blastocyst rate, decreased hatching rate and increased apoptosis in blastocysts. In conclusion, the present study demonstrates that repeated superovulation adversely affects mouse oocyte quality by altering the distribution of cytoplasmic organelles, increasing oxidative stress and decreasing Oct4 expression, resulting in poor developmental potential of the embryos.

DGKι regulates presynaptic release during mGluR-dependent LTD.

Diacylglycerol (DAG) is an important lipid second messenger. DAG signalling is terminated by conversion of DAG to phosphatidic acid (PA) by diacylglycerol kinases (DGKs). The neuronal synapse is a major site of DAG production and action; however, how DGKs are targeted to subcellular sites of DAG generation is largely unknown. We report here that postsynaptic density (PSD)-95 family proteins interact with and promote synaptic localization of DGKι. In addition, we establish that DGKι acts presynaptically, a function that contrasts with the known postsynaptic function of DGKζ, a close relative of DGKι. Deficiency of DGKι in mice does not affect dendritic spines, but leads to a small increase in presynaptic release probability. In addition, DGKι-/- synapses show a reduction in metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD) at neonatal (∼2 weeks) stages that involve suppression of a decrease in presynaptic release probability. Inhibition of protein kinase C normalizes presynaptic release probability and mGluR-LTD at DGKι-/- synapses. These results suggest that DGKι requires PSD-95 family proteins for synaptic localization and regulates presynaptic DAG signalling and neurotransmitter release during mGluR-LTD.

Oocytes recovered after ovarian tissue slow freezing have impaired H2AX phosphorylation and functional competence.

It has been shown that oocytes isolated from ovarian tissue cryopreservation acquire DNA damage during the process of freeze-thawing. Using a mouse model, here we have investigated the functional competence and phosphorylation of H2AX (γ-H2AX) in germinal vesicle (GV) and parthenogenetically activated oocytes derived from conventional ovarian tissue slow freezing and vitrification techniques. The number of GV-stage oocytes with γ-H2AX foci was not significantly different between the slow-freezing and vitrification groups. Although the in vitro maturation (IVM) potential of GV oocytes in the slow-freezing group showed a significant delay (P<0.0001) in the process of germinal vesicle breakdown, no difference in the maturation rate was observed between the two protocols. Nevertheless, parthenogenetic activation of IVM oocytes using strontium chloride showed a significantly lower activation rate in the slow-freezing group compared with the vitrification (P<0.05) and control (P<0.01) groups. Importantly, H2AX phosphorylation was significantly perturbed in the slow-freezing group in comparison to the control (P<0.05). Therefore, we conclude that impaired sensing of DNA strand breaks and repair processes are associated with the reduced functional competence of the oocytes recovered from the slow-freezing group, which may have a significant impact on the reproductive outcome.

Methyl parathion inhibits the nuclear maturation, decreases the cytoplasmic quality in oocytes and alters the developmental potential of embryos of Swiss albino mice.

Methyl parathion (MP) is one of the most commonly used and extremely toxic organophosphorous group of pesticide. A large number of studies in the literature suggest that it has adverse effects on the male reproductive system. However, there is limited information about its toxicity to the female reproductive system. In the present study we report the toxic effects of methyl parathion on the female reproductive system using Swiss albino mice as the experimental model. The female mice were administered orally with 5, 10 and 20mg/kg of MP. One week later, the mice were superovulated with pregnant mare serum gonadotrophin (PMSG) and human chorionic gonadotrophin (hCG) to study the quality of the oocytes, spindle organization, developmental potential of early embryos and the DNA integrity in blastocysts. MP exposure resulted in a non-significant decrease in the number of primordial follicles and increased DNA damage in granulosa cells. Though MP did not have any effect on the ovulation it had a significant inhibitory effect on the nuclear maturity of oocytes which was associated with spindle deformity. In addition, the oocytes had higher cytoplasmic abnormalities with depleted glutathione level. Even though it did not have any effect on the fertilization and blastocyst rate at lower doses, at 20 mg/kg MP it resulted in a significant decrease in blastocyst hatching, decrease in cell number and high DNA damage. While low body weight gain was observed in F1 generation from 5mg/kg group, at higher dose, the body weight in F1 generation was marginally higher than control. Post-natal death in F1 generation was observed only in mice treated with 20mg/kg MP. In conclusion, we report that MP has adverse effects on the oocyte quality, developmental potential of the embryo and reproductive outcome.

Strategies for developing 3D printed ovarian model for restoring fertility.

Ovaries play a crucial role in the regulation of numerous essential processes that occur within the intricate framework of female physiology. They are entrusted with the responsibility of both generating a new life and orchestrating a delicate hormonal symphony. Understanding their functioning is crucial for gaining insight into the complexities of reproduction, health, and fertility. In addition, ovaries secrete hormones that are crucial for both secondary sexual characteristics and the maintenance of overall health. A three-dimensional (3D) prosthetic ovary has the potential to restore ovarian function and preserve fertility in younger females who have undergone ovariectomies or are afflicted with ovarian malfunction. Clinical studies have not yet commenced, and the production of 3D ovarian tissue for human implantation is still in the research phase. The main challenges faced while creating a 3D ovary for in vivo implantation include sustenance of ovarian follicles, achieving vascular infiltration into the host tissue, and restoring hormone circulation. The complex ovarian microenvironment that is compartmentalized and rigid makes the biomimicking of the 3D ovary challenging in terms of biomaterial selection and bioink composition. The successful restoration of these properties in animal models has led to expectations for the development of human ovaries for implantation. This review article summarizes and evaluates the optimal 3D models of ovarian structures and their safety and efficacy concerns to provide concrete suggestions for future research.

Dual Checkpoint Aptamer Immunotherapy: Unveiling Tailored Cancer Treatment Targeting CTLA-4 and NKG2A.

Recent strides in immunotherapy have illuminated the crucial role of CTLA-4 and PD-1/PD-L1 pathways in contemporary oncology, presenting both promises and challenges in response rates and adverse effects. This study employs a computational biology tool (in silico approach) to craft aptamers capable of binding to dual receptors, namely, inhibitory CTLA4 and NKG2A, thereby unleashing both T and NK cells and enhancing CD8+ T and NK cell functions for tumor cell lysis. Computational analysis highlighted AYA22T-R2-13 with HADDOCK scores of -78.2 ± 10.2 (with CTLA4), -60.0 ± 4.2 (with NKG2A), and -77.5 ± 5.6 (with CD94/NKG2A). Confirmation of aptamer binding to targeted proteins was attained via ELISA and flow cytometry methods. In vitro biological functionality was assessed using lactate dehydrogenase (LDH) cytotoxicity assay. Direct and competitive assays using ELISA and flow cytometry demonstrated the selective binding of AYA22T-R2-13 to CTLA4 and NKG2A proteins, as well as to the cell surface receptors of IL-2-stimulated T cells and NK cells. This binding was inhibited in the presence of competition from CTLA4 or NKG2A proteins. Remarkably, the blockade of CTLA4 or NKG2A by AYA22T-R2-13 augmented human CD8 T cell- and NK cell-mediated tumor cell lysis in vitro. Our findings highlight the precise binding specificity of AYA22T-R2-13 for CTLA4-B7-1/B7-2 (CD80/CD86) or CD94/NKG2A-HLA-E interactions, positioning it as a valuable tool for immune checkpoint blockade aptamer research in murine tumor models. These in vitro studies establish a promising foundation for further enhancing binding capacity and establishing efficacy and safety in animal models. Consequently, our results underscore the potential of AYA22T-R2-13 in cancer immunotherapy, offering high specificity, low toxicity, and the potential for cost-effective production.

Novel Vpx virus-like particles to improve cytarabine treatment response against acute myeloid leukemia.

Knowledge of the molecular pathogenesis of acute myeloid leukemia has advanced in recent years. Despite novel treatment options, acute myeloid leukemia remains a survival challenge for elderly patients. We have recently shown that the triphosphohydrolase SAMHD1 is one of the factors determining resistance to Ara-C treatment. Here, we designed and tested novel and simpler virus-like particles incorporating the lentiviral protein Vpx to efficiently and transiently degrade SAMHD1 and increase the efficacy of Ara-C treatment. The addition of minute amounts of lentiviral Rev protein during production enhanced the generation of virus-like particles. In addition, we found that our 2nd generation of virus-like particles efficiently targeted and degraded SAMHD1 in AML cell lines with high levels of SAMHD1, thereby increasing Ara-CTP levels and response to Ara-C treatment. Primary AML blasts were generally less responsive to VLP treatment. In summary, we have been able to generate novel and simpler virus-like particles that can efficiently deliver Vpx to target cells.

New High-Affinity Thrombin Aptamers for Advancing Coagulation Therapy: Balancing Thrombin Inhibition for Clot Prevention and Effective Bleeding Management with Antidote.

Thrombin is a key enzyme involved in blood clotting, and its dysregulation can lead to thrombotic diseases such as stroke, myocardial infarction, and deep vein thrombosis. Thrombin aptamers have the potential to be used as therapeutic agents to prevent or treat thrombotic diseases. Thrombin DNA aptamers developed in our laboratory exhibit high affinity and specificity to thrombin. In vitro assays have demonstrated their efficacy by significantly decreasing Factor II activity and increasing PT and APTT times in both plasma and whole blood. Aptamers AYA1809002 and AYA1809004, the two most potent aptamers, exhibit high affinity for their target, with affinity constants (Kd) of 10 nM and 13 nM, respectively. Furthermore, the in vitro activity of these aptamers displays dose-dependent behavior, highlighting their efficacy in a concentration-dependent manner. In vitro stability assessments reveal that the aptamers remain stable in plasma and whole blood for up to 24 h. This finding is crucial for their potential application in clinical settings. Importantly, the thrombin inhibitory activity of the aptamers can be reversed by employing reverse complement sequences, providing a mechanism to counteract their anticoagulant effects when necessary to avoid excessive bleeding. These thrombin aptamers have been determined to be safe, with no observed mutagenic or immunogenic effects. Overall, these findings highlight the promising characteristics of these newly developed thrombin DNA aptamers, emphasizing their potential for therapeutic applications in the field of anticoagulation therapy. Moreover, the inclusion of an antidote in the coagulation therapy regimen can improve patient safety, ensure greater therapeutic efficacy, and minimize risk during emergency situations.

Haploid Parthenogenetic Embryos Exhibit Unique Stress Response to pH, Osmotic and Oxidative Stress.

Preimplantation-stage embryos are susceptible to various types of stress when cultured in vitro. Parthenogenetic embryos that lack spermatozoa contribution exhibit aberrant developmental dynamics due to their uniparental origin. Herein, we assessed whether the absence of paternal genome affects the susceptibility of the embryos to pH, osmotic and oxidative stress. Haploid parthenogenetic embryos (HPE) (activated oocytes with 1 pronucleus and 2 polar bodies) were generated by incubating cumulus oocyte complexes of Swiss albino mice with 10 mM strontium chloride for 3 h. Normally fertilized embryos (NFE) (fertilized oocytes with 2 pronuclei and 2 polar bodies) were derived using in vitro fertilization. At 2-cell stage, both HPE and NFE were exposed to various stressors including pH (6.8 to 8.2), osmotic (isotonic, hypotonic, and hypertonic), and peroxidatic oxidative (H2O2, 25 µM) stress. Endoplasmic reticulum stress response, mitochondrial membrane potential, and the rate of blastocyst development were assessed. HPE were susceptible to alteration in the pH that was well tolerated by NFE. Similarly, HPE displayed remarkable difference in sensitivity to hypertonic stress and oxidative stress compared to NFE. The results clearly indicate that the oocytes that develop into embryos in the absence of paternal contribution are more vulnerable to environmental stressors, further highlighting the importance of spermatozoa contribution and/or the ploidy status in mitigating these stressors and towards healthy early embryo development.

Expanding the phenotypic spectrum of Chromosome 16p13.11 microduplication: A multicentric analysis of 206 patients.

INTRODUCTION: Recurrent chromosome 16p13.11 microduplication has been characterised in the literature as a cause of developmental delay, learning difficulties and behavioural abnormalities. It is a neurosusceptibility locus and has incomplete penetrance and variable expression. Other clinical features, such as cardiac abnormalities have also been reported. The duplicated region contains the MYH11 gene, which encodes the protein myosin-11 and is a component of the myosin heavy chain in smooth muscle. Recent literature has suggested 16p13.11 microduplication as one of the possible risk factors for thoracic aortic aneurysms and dissection (TAAD). Therefore, we studied the detailed phenotype of cases of chromosome 16p13.11 microduplication from seven centres in the United Kingdom (UK) to expand the phenotype, focusing on the cardiac abnormalities. METHODS: All individuals with a chromosome 16p13.11 microduplication seen in Clinical Genetics prior to June 2017 in 6 centres (prior to 2018 in the seventh centre) were identified through the regional genetics laboratory databases. A Microsoft Excel® proforma was created and clinical data was collected retrospectively from clinical genetics databases from the seven genetics services in the UK. The data was collated and analysed collectively. RESULTS: The majority of the individuals presented with (72%) developmental delay and (62%) behavioural abnormalities, in keeping with the published literature. 27% had some dysmorphic features, 14% had visual impairment and 8% had congenital cardiac abnormalities. Echocardiograms were performed in 50% of patients, and only 3.8% patients had aortic dilatation and no one had aortic dissection. 9.7% of patients were found to have a second genetic/chromosomal diagnosis, especially where there were additional phenotypic features. CONCLUSION: 16p13.11 microduplication is a neurosusceptibility locus and is associated with variable expression. It may be helpful to refer children with 16p13.11 microduplication for a cardiac review for congenital cardiac abnormalities and also for ophthalmological assessment. Further prospective studies with cardiac assessments are recommended in this cohort of patients to determine whether ongoing aortic surveillance is indicated. Guidelines about the frequency of surveillance are indicated, especially in individuals with normal cardiac findings. We also highlight the importance of considering a second diagnosis if the phenotype is inconsistent with that reported.

Probing the functional equivalence of otoferlin and synaptotagmin 1 in exocytosis.

Cochlear inner hair cells (IHCs) use Ca(2+)-dependent exocytosis of glutamate to signal sound information. Otoferlin (Otof), a C(2) domain protein essential for IHC exocytosis and hearing, may serve as a Ca(2+) sensor in vesicle fusion in IHCs that seem to lack the classical neuronal Ca(2+) sensors synaptotagmin 1 (Syt1) and Syt2. Support for the Ca(2+) sensor of fusion hypothesis for otoferlin function comes from biochemical experiments, but additional roles in late exocytosis upstream of fusion have been indicated by physiological studies. Here, we tested the functional equivalence of otoferlin and Syt1 in three neurosecretory model systems: auditory IHCs, adrenal chromaffin cells, and hippocampal neurons. Long-term and short-term ectopic expression of Syt1 in IHCs of Otof (-/-) mice by viral gene transfer in the embryonic inner ear and organotypic culture failed to rescue their Ca(2+) influx-triggered exocytosis. Conversely, virally mediated overexpression of otoferlin did not restore phasic exocytosis in Syt1-deficient chromaffin cells or neurons but enhanced asynchronous release in the latter. We further tested exocytosis in Otof (-/-) hippocampal neurons and in Syt1(-/-) IHCs but found no deficits in vesicle fusion. Expression analysis of different synaptotagmin isoforms indicated that Syt1 and Syt2 are absent from mature IHCs. Our data argue against a simple functional equivalence of the two C(2) domain proteins in exocytosis of IHC ribbon synapses, chromaffin cells, and hippocampal synapses.

New strategies to treat AML: novel insights into AML survival pathways and combination therapies.

The effective treatment of acute myeloid leukemia (AML) is very challenging. Due to the immense heterogeneity of this disease, treating it using a "one size fits all" approach is ineffective and only benefits a subset of patients. Instead, there is a shift towards more personalized treatment based on the patients' genomic signature. This shift has facilitated the increased revelation of novel insights into pathways that lead to the survival and propagation of AML cells. These AML survival pathways are involved in drug resistance, evasion of the immune system, reprogramming metabolism, and impairing differentiation. In addition, based on the reports of enhanced clinical efficiencies when combining drugs or treatments, deeper investigation into possible pathways, which can be targeted together to increase treatment response in a wider group of patients, is warranted. In this review, not only is a comprehensive summary of targets involved in these pathways provided, but also insights into the potential of targeting these molecules in combination therapy will be discussed.

Anaerobic ammonia-oxidizing bacteria in tropical bioaugmented zero water exchange aquaculture ponds.

Bioaugmented zero water exchange aquaculture production systems (ZWEAPS) maintained with minimal or no water exchange prevent the ammonia accumulation in the system, leading to environmental sustainability and biosecurity. The microbes in the bioaugmented ZWEAPS plays a major role in maintaining low levels of ammonia through ammonia oxidation and nitrite oxidation. The comprehensive understanding on anammox population in the systems will provide an insight on the environmental factors controlling the functional anammox bacterial communities for potential biostimulation and augmented ammonia removal in ZWEAPS. The sediment metagenome of such three tropical bioaugmented ZWE shrimp culture ponds were analysed to determine the diversity, distribution and abundance of anaerobic ammonia-oxidizing (anammox) bacteria based on hydrazine oxidoreductase (hzo) gene as a phylogenetic marker. The restriction fragment length polymorphism (RFLP) phylotypes from the clone libraries were identified with maximum distribution to Candidatus Kuenenia, as the dominant population in the study sites with high ammonia load followed by Candidatus Scalindua. The environmental factors associated with the abundance and diversity of the anammox population were analysed using RDA and Pearson correlation. The samples of final culturing period (75th day) of TCR-S ZWE pond was observed with the highest operational taxonomic unit (OTU)-based diversity, where comparatively higher ammonia (water 0.71 mg L-1 and sediment 1.21 mg L-1) was recorded among the study sites. The gene abundance of the anammox population ranged from 106 to 107 copies per gram of sediment, in spite of less diversity. The physiochemical factors such as ammonia, nitrite, redox potential and the total organic carbon indicated a strong and positive correlation to the abundance and distribution of the anammox population, which highlights the importance of anammox communities and the potential of biostimulation for ammonia removal in the aquaculture systems.

Computational analysis of successional changes in the microbial population and community diversity of the immobilized marine nitrifying bacterial consortium in a nitrifying packed bed bioreactor.

Nitrifying bioreactor (NBR) connected to the recirculating aquaculture system (RAS), has a greater emphasis on the biological treatment of wastewater. Nitrifying bacterial consortium (NBC) formed bio-film on the substratum activating the NBR, and it was observed with high nitrification potential in shrimp maturation systems. Scanning Electron Microscopy (SEM) revealed the integrity of the biofilm substantiated with biomineralization. The fate of the matured bio-film population on subsequent operation under RAS, and the aggregated population at different points of RAS, including the rearing water were determined using fingerprints of Denaturing Gradient Gel Electrophoresis (DGGE). Altogether, 38 OTUs of biofilm sample and 35 OTUs of water samples represented the bacterial communities; the shared and unique OTUs indicated the diversity of the population at different time intervals in the operation of the NBR. The mathematical (range-weighted richness) and statistical (diversity indices) interpretation unveiled the OTUs based high bacterial diversity in the biofilm supporting the compositional changes and determined the distance between the community cluster. Ordination analyses indicated the population shift and stability of the activated bio-film till the matured biofilm community got established in the RAS. The DGGE with mathematical and statistical analysis revealed microbial diversity (high Shannon index, species richness and evenness), abundance (relative intensity), consecutive change in the population composition (OTUs, Rr index), and the dynamics (Δt) in the system during the operation.