A novel function for CDK2 activity at meiotic crossover sites.

Genetic diversity in offspring is induced by meiotic recombination, which is initiated between homologs at >200 sites originating from meiotic double-strand breaks (DSBs). Of this initial pool, only 1-2 DSBs per homolog pair will be designated to form meiotic crossovers (COs), where reciprocal genetic exchange occurs between parental chromosomes. Cyclin-dependent kinase 2 (CDK2) is known to localize to so-called "late recombination nodules" (LRNs) marking incipient CO sites. However, the role of CDK2 kinase activity in the process of CO formation remains uncertain. Here, we describe the phenotype of 2 Cdk2 point mutants with elevated or decreased activity, respectively. Elevated CDK2 activity was associated with increased numbers of LRN-associated proteins, including CDK2 itself and the MutL homolog 1 (MLH1) component of the MutLγ complex, but did not lead to increased numbers of COs. In contrast, reduced CDK2 activity leads to the complete absence of CO formation during meiotic prophase I. Our data suggest an important role for CDK2 in regulating MLH1 focus numbers and that the activity of this kinase is a key regulatory factor in the formation of meiotic COs.

CDK10 Mutations in Humans and Mice Cause Severe Growth Retardation, Spine Malformations, and Developmental Delays.

In five separate families, we identified nine individuals affected by a previously unidentified syndrome characterized by growth retardation, spine malformation, facial dysmorphisms, and developmental delays. Using homozygosity mapping, array CGH, and exome sequencing, we uncovered bi-allelic loss-of-function CDK10 mutations segregating with this disease. CDK10 is a protein kinase that partners with cyclin M to phosphorylate substrates such as ETS2 and PKN2 in order to modulate cellular growth. To validate and model the pathogenicity of these CDK10 germline mutations, we generated conditional-knockout mice. Homozygous Cdk10-knockout mice died postnatally with severe growth retardation, skeletal defects, and kidney and lung abnormalities, symptoms that partly resemble the disease's effect in humans. Fibroblasts derived from affected individuals and Cdk10-knockout mouse embryonic fibroblasts (MEFs) proliferated normally; however, Cdk10-knockout MEFs developed longer cilia. Comparative transcriptomic analysis of mutant and wild-type mouse organs revealed lipid metabolic changes consistent with growth impairment and altered ciliogenesis in the absence of CDK10. Our results document the CDK10 loss-of-function phenotype and point to a function for CDK10 in transducing signals received at the primary cilia to sustain embryonic and postnatal development.

Nerve Conduction Abnormalities in Pre-Diabetics and Asymptomatic Diabetics.

Aim: To determine the electrophysiological abnormalities in pre-diabetics and/ or asymptomatic diabetics and analyse the role of nerve conduction for recognizing distal symmetric polyneuropathy. Material and Methods: A total of 180 subjects were categorized as: Group A: healthy Subjects (n=60), Group B Pre-diabetics (IFG +IGT, n=60) and Group C: Asymptomatic type 2 diabetics (n=60). Results: Electrophysiological studies revealed that amplitude of B/L Sural SNAP and tibial CMAP was significantly lower in affected pre-diabetics and asymptomatic diabetics. The presence of significant f wave latency was also noted in both these groups, more among asymptomatic diabetics. The observations on distal latency and nerve conduction velocity of sensory and motor nerves were statistically nonsignificant. Conclusion: Sensory nerve abnormality was more obvious than motor nerve abnormality in the pre-diabetic subjects. The changes in amplitude of motor nerve abnormality was observed late in course of disease i.e. in asymptomatic diabetic group than pre-diabetics. The amplitude of sensory nerve action potential and F wave latency parameters were the most sensitive measures of peripheral neuropathy in early diabetics in our study.

Systemic Wegener's granulomatosis with severe oculo-otological manifestations.

A case of Granulomatosis with Polyangiitis (Wegener's) is described in which the common presenting symptoms were hoarseness, deafness and ocular manifestations including marked redness and congestion of both eyes. The case findings and other systemic involvements are reviewed briefly along with histological and immunological discussions.

Noise-induced hormonal & morphological malformations in breeding pigeons.

Environmental pollution has the potential to have a significant impact on animal's health especially on birds due to daily exposure and habitat. This experimental study was carried out for a 60 days period in which, a total of 24 pigeon birds with suitable weight (80-100 g) were kept in Animal house with suitable environmental conditions viz, controlled temperature, humidity & light source to minimize any other stress. Out of twenty-four, eighteen birds were divided into three treatment groups (6 birds in each group). Whole experiment was run in triplicate manner in breeding season. One served as Control (Group 1) and remaining three were experimental groups including Road traffic noise (Group 2), Military noise (Group 3) & Human activities noise (Group 4). Noise was applied as recorded high intensity music (1125 Hz/ 90 dB) through speakers for 5-6 hrs. daily. Blood sampling was done after 20, 40 and 60 days by sacrificing treatment birds. Noise stress significantly (p<0.05) increase the serum levels of corticosterone and thyroid stimulating hormone (TSH) in Group 2 while significantly (p<0.05) decrease the serum levels of luteinizing hormone (LH) and follicle stimulating hormone (FSH) of Group 3 birds. Moreover, major fault bars formation was seen both in Group 2 and Group 3. It was concluded as that Noise stress caused rise in serum levels of Corticosterone and TSH but fall in LH and FSH. Along with fault bars formation was also prominent in all treatment groups due to stress hormone.

Identification PMS1 and PMS2 as potential meiotic substrates of CDK2 activity.

Cyclin dependent-kinase 2 (CDK2) plays important functions during the mitotic cell cycle and also facilitates several key events during germ cell development. The majority of CDK2's known meiotic functions occur during prophase of the first meiotic division. Here, CDK2 is involved in the regulation of meiotic transcription, the pairing of homologous chromosomes, and the maturation of meiotic crossover sites. Despite that some of the CDK2 substrates are known, few of them display functions in meiosis. Here, we investigate potential meiotic CDK2 substrates using in silico and in vitro approaches. We find that CDK2 phosphorylates PMS2 at Thr337, PMS1 at Thr331, and MLH1 in vitro. Phosphorylation of PMS2 affects its interaction with MLH1 to some degree. In testis extracts from mice lacking Cdk2, there are changes in expression of PMS2, MSH2, and HEI10, which may be reflective of the loss of CDK2 phosphorylation. Our work has uncovered a few CDK2 substrates with meiotic functions, which will have to be verified in vivo. A better understanding of the CDK2 substrates will help us to gain deeper insight into the functions of this universal kinase.

The Use of Dog Collars Offers Significant Benefits to Rabies Vaccination Campaigns: The Case of Zanzibar, Tanzania.

Tools and resources that could increase dog vaccination coverage have become increasingly critical towards progressing the goal to eliminate dog-mediated human rabies by 2030. In this regard, dog collars that are fitted during vaccination campaigns could potentially enhance owner participation. The use of dog collars will, however, increase the cost per dog vaccinated and the impact and benefit of this practice should be elucidated. This study evaluated the impact of dog collars by testing the perception and related behavioural influences in communities in Zanzibar. In this cross-sectional investigation-conducted approximately two months after the implementation of a mass dog vaccination (MDV) where dog collars were provided to vaccinated dogs-data were collected from 600 respondents in 56 municipal wards in Zanzibar. Descriptive analyses and logistic regressions were undertaken to determine the impact the collars had on respondents with regards to (i) engaging with the community dogs, (ii) health seeking behaviour after exposure, and (iii) overall participation during dog vaccination campaigns. From the data, it was evident that the collars had a positive impact on the community's perception of dogs, with 57% of the respondents feeling safer around a dog with a collar, while 66% of the respondents felt less safe around a dog without a collar. Furthermore, the collars had a positive impact on participation during dog vaccination campaigns. Of the 142 respondents who owned dogs, 64% reported that the collars made them more likely to take their dogs for vaccination, and 95% felt that the collar was an important sign of the dog's vaccination status. This study demonstrated that dog collars could not only improve participation during dog vaccination campaigns, but that they could also play a significant role in the community's perception of rabies vaccination campaigns and vaccinated dogs in general.

Occurrence of thermophilic Campylobacter spp. contamination on vegetable farms in Malaysia.

The aim of the present study was to examine the prevalence of thermophilic Campylobacter spp. (Campylobacter jejuni and Campylobacter coli) in soil, poultry manure, irrigation water, and freshly harvested vegetables from vegetable farms in Malaysia. C. jejuni was detected in 30.4% and 2.7% of the soil samples, 57.1% and 0% of the manure samples, and 18.8% and 3% of the vegetable samples from farm A and farm B, respectively, when using the MPNPCR method. Campylobacter spp. was not found in any of the irrigation water samples tested. Therefore, the present results indicate that the aged manure used by farm A was more contaminated than the composted manure used by farm B. Mostly, the leafy and root vegetables were contaminated. C. coli was not detected in any of the samples tested in the current study. Both farms tested in this study were found to be contaminated by campylobacters, thereby posing a potential risk for raw vegetable consumption in Malaysia. The present results also provide baseline data on Campylobacter contamination at the farm level.

Diverse roles for CDK-associated activity during spermatogenesis.

The primary function of cyclin-dependent kinases (CDKs) in complex with their activating cyclin partners is to promote mitotic division in somatic cells. This canonical cell cycle-associated activity is also crucial for fertility as it allows the proliferation and differentiation of stem cells within the reproductive organs to generate meiotically competent cells. Intriguingly, several CDKs exhibit meiosis-specific functions and are essential for the completion of the two reductional meiotic divisions required to generate haploid gametes. These meiosis-specific functions are mediated by both known CDK/cyclin complexes and meiosis-specific CDK-regulators and are important for a variety of processes during meiotic prophase. The majority of meiotic defects observed upon deletion of these proteins occur during the extended prophase I of the first meiotic division. Importantly a lack of redundancy is seen within the meiotic arrest phenotypes described for many of these proteins, suggesting intricate layers of cell cycle control are required for normal meiotic progression. Using the process of male germ cell development (spermatogenesis) as a reference, this review seeks to highlight the diverse roles of selected CDKs their activators, and their regulators during gametogenesis.

CDK2 regulates the NRF1/Ehmt1 axis during meiotic prophase I.

Meiosis generates four genetically distinct haploid gametes over the course of two reductional cell divisions. Meiotic divisions are characterized by the coordinated deposition and removal of various epigenetic marks. Here we propose that nuclear respiratory factor 1 (NRF1) regulates transcription of euchromatic histone methyltransferase 1 (EHMT1) to ensure normal patterns of H3K9 methylation during meiotic prophase I. We demonstrate that cyclin-dependent kinase (CDK2) can bind to the promoters of a number of genes in male germ cells including that of Ehmt1 through interaction with the NRF1 transcription factor. Our data indicate that CDK2-mediated phosphorylation of NRF1 can occur at two distinct serine residues and negatively regulates NRF1 DNA binding activity in vitro. Furthermore, induced deletion of Cdk2 in spermatocytes results in increased expression of many NRF1 target genes including Ehmt1 We hypothesize that the regulation of NRF1 transcriptional activity by CDK2 may allow the modulation of Ehmt1 expression, therefore controlling the dynamic methylation of H3K9 during meiotic prophase.

Discovery of a chemical probe for PRDM9.

PRDM9 is a PR domain containing protein which trimethylates histone 3 on lysine 4 and 36. Its normal expression is restricted to germ cells and attenuation of its activity results in altered meiotic gene transcription, impairment of double-stranded breaks and pairing between homologous chromosomes. There is growing evidence for a role of aberrant expression of PRDM9 in oncogenesis and genome instability. Here we report the discovery of MRK-740, a potent (IC50: 80 ± 16 nM), selective and cell-active PRDM9 inhibitor (Chemical Probe). MRK-740 binds in the substrate-binding pocket, with unusually extensive interactions with the cofactor S-adenosylmethionine (SAM), conferring SAM-dependent substrate-competitive inhibition. In cells, MRK-740 specifically and directly inhibits H3K4 methylation at endogenous PRDM9 target loci, whereas the closely related inactive control compound, MRK-740-NC, does not. The discovery of MRK-740 as a chemical probe for the PRDM subfamily of methyltransferases highlights the potential for exploiting SAM in targeting SAM-dependent methyltransferases.

Theoretical design of metal-phthalocyanine dye-sensitized solar cells with improved efficiency.

The present work carried out a theoretical study of the electronic structures, absorption spectra, and photovoltaic performance of two series of transition metal-phthalocyanine derived from nonperipheral electron-donating substituents, either (2-phenyl) phenoxy(M-PC1) or quinoleinoxy(M-PC2). The DFT and TD-DFT were employed for this study. The effect of modifying the central metal atoms and the substitution on cell performance were investigated in terms of polarizability (α), hyper-polarizability (ß), chemical potential (µ), chemical hardness (η), electrophilicity power (ω), FMOs, energy gaps, UV/vis absorption spectra and injected driving force (ΔGinject), light harvesting efficiencies (LHE), total reorganization energy (λtot), open circuit photovoltage (Voc), and life time of the excited state (τ). The results obtained by using these parameters showed that the replacement of (2-phenyl) phenoxy by a proposed substituent such as quinoleinoxy would increase the hyper-polarizability, light harvesting efficiency, and open circuit photovoltage, while on the other hand the reorganization energy and the injection driving force are decreased. Modifying central metal atoms, such as Zn, Cd, Pd, and Pt, exhibited good performance in terms of the driving force of electron injection, charge transfer characteristics, and dye reorganization as compared with the Cu reference dye. The findings provided a useful prediction and perspective for the promising future for high-efficiency dye-sensitized solar cells (DSSCs) with dyes based on phthalocyanine. Graphical abstract Photovoltaic performance of Metallo-phthalocyanine contening (2-phenyl) phenoxy and quinoleinoxy.

Metabolic Remodeling during Liver Regeneration.

Liver disease is linked to a decreased capacity of hepatocytes to divide. In addition, cellular metabolism is important for tissue homeostasis and regeneration. Since metabolic changes are a hallmark of liver disease, we investigated the connections between metabolism and cell division. We determined global metabolic changes at different stages of liver regeneration using a combination of integrated transcriptomic and metabolomic analyses with advanced functional redox in vivo imaging. Our data indicate that blocking hepatocyte division during regeneration leads to mitochondrial dysfunction and downregulation of oxidative pathways. This resulted in an increased redox ratio and hyperactivity of alanine transaminase allowing the production of alanine and α-ketoglutarate from pyruvate when mitochondrial functions are impaired. Our data suggests that during liver regeneration, cell division leads to hepatic metabolic remodeling. Moreover, we demonstrate that hepatocytes are equipped with a flexible metabolic machinery able to adapt dynamically to changes during tissue regeneration.

MAGE-1-specific precursor cytotoxic T-lymphocytes present among tumor-infiltrating lymphocytes from a patient with breast cancer: characterization and antigen-specific activation.

A potential target for development of tumor-specific immunotherapeutic strategies is the MAGE-1 gene. We have utilized a recently developed recombinant canarypox (ALVAC) virus vector containing the MAGE-1 gene (vCP235) to activate CTLs from a breast cancer patient bearing a MAGE-1+ tumor. Tumor-infiltrating lymphocytes (TILs) obtained from the tumor of a patient were stimulated in vitro with irradiated autologous peripheral blood mononuclear cells acutely infected with the vCP235 construct. These TILs preferentially expanded approximately 6-fold over a 16-day culture period and specifically recognized an allogeneic transformed B-cell line acutely infected with a vaccinia-MAGE-1 recombinant targeting vector (vP1188) in the context of HLA-A2 and/or B7. TCR V beta analysis of in vitro expanded T cells by a quantitative multiprobe RNase protection assay revealed preferential expansion of TCR V beta 6.3 and V beta 6.4. In addition, homologous T-cell receptor beta CDR3 joining sequences were found in the in vitro stimulated cultures. These results suggest that tumor antigen-specific, MHC-restricted CTLs may be derived from precursor CTLs present in TILs obtained from patients with MAGE-1+ tumors by in vitro stimulation with recombinant avipox MAGE-1 virus-infected autologous cells. Collectively, these findings provide a rationale for tumor-associated antigen-based immunization as a means of activating precursor CTLs residing in patients with tumors expressing defined tumor-associated antigens such as MAGE-1.

The Indispensable Role of Cyclin-Dependent Kinase 1 in Skeletal Development.

Skeletal development is tightly regulated through the processes of chondrocyte proliferation and differentiation. Although the involvement of transcription and growth factors on the regulation of skeletal development has been extensively studied, the role of cell cycle regulatory proteins in this process remains elusive. To date, through cell-specific loss-of-function experiments in vivo, no cell cycle regulatory proteins have yet been conclusively shown to regulate skeletal development. Here, we demonstrate that cyclin-dependent kinase 1 (Cdk1) regulates skeletal development based on chondrocyte-specific loss-of-function experiments performed in a mouse model. Cdk1 is highly expressed in columnar proliferative chondrocytes and is greatly downregulated upon differentiation into hypertrophic chondrocytes. Cdk1 is essential for proper chondrocyte proliferation and deletion of Cdk1 resulted in accelerated differentiation of chondrocytes. In vitro and ex vivo analyses revealed that Cdk1 is an essential cell cycle regulatory protein for parathyroid hormone-related peptide (PTHrP) signaling pathway, which is critical to chondrocyte proliferation and differentiation. These results demonstrate that Cdk1 functions as a molecular switch from proliferation to hypertrophic differentiation of chondrocytes and thus is indispensable for skeletal development. Given the availability of inhibitors of Cdk1 activity, our results could provide insight for the treatment of diseases involving abnormal chondrocyte proliferation, such as osteoarthritis.

Model concept for nitrate and nitrite utilization during anoxic transformation in the bulk water phase of municipal wastewater under sewer conditions.

A two-stage anoxic transformation process, involving growth of biomass utilizing two types of different electron acceptors, namely nitrate and nitrite, has been observed. The present water quality modules established for sewer processes cannot account for the two-stage process. This paper outlines the development of a model concept that enables the two-stage anoxic transformation process to be simulated. The proposed model is formulated in a matrix form that is similar to the Activated Sludge Models and Sewer Process Model matrices. The model was successfully applied to simulate changes in nitrate and nitrite concentrations during anoxic transformations in the bulkwater phase of municipal wastewater.

Cloning and expression in Escherichia coli of a synthetic gene encoding the extracellular domain of the human muscle acetylcholine receptor alpha-subunit.

To better define the antigenic sites on the human muscle acetylcholine receptor (AChR) that are involved in stimulating the production of pathogenic antibodies in myasthenia gravis (MG), the nucleotide sequence encoding the major extracellular domain of the AChR alpha subunit was chemically synthesized. The gene cassettes encoding amino acids (aa) 1-85 (AChR-I) and 86-210 (AChR-II), were cloned individually, and the coding sequence representing the complete major extracellular domain (aa 1-210; AChR-C) was obtained by subsequent fusion of cassettes encoding AChR-I and AChR-II. The genes were inserted into the inducible expression plasmid, pKK-223-3, and expressed in vitro and in vivo in Escherichia coli. Biological activity was demonstrated by immunoprecipitation of in vitro-synthesized AChR-C by sera from MG patients and by the alpha-bungarotoxin-binding activity of E. coli-synthesized AChR-II and AChR-C. The availability of the recombinant AChR polypeptides should facilitate studies on the molecular basis of the autoimmune response in MG.

Ligand-mediated cytolysis of tumor cells: use of heregulin-zeta chimeras to redirect cytotoxic T lymphocytes.

New specificities may be engrafted onto lymphocytes by the transfer of genes for chimeric receptors that combine antigen recognition and signal-transducing elements. We have engineered and evaluated a new class of chimeric receptors that use the natural ligands of receptors found to be frequently overexpressed by cancer cells. The heregulin molecule, a ligand for Her3 and Her4 receptors when fused with the CD3 zeta-chain, was capable of redirecting T lymphocytes to recognize and respond to cancer cell lines that overexpress these receptors. Thus, CD8+ T lymphocytes were isolated from a healthy individual and transduced to express the chimeric heregulin-zeta receptor. These modified effector cells acquired the ability to specifically lyse a breast cancer cell line that overexpresses Her3 and Her4. A new class of chimeric receptors, such as heregulin-zeta, endowing anti-cancer effector cells with the potential to recognize and eliminate tumor targets, are likely to increase the effectiveness of adoptive immunotherapy for the treatment of cancer.

Integrated design of sewers and wastewater treatment plants.

Sewer system design must be integrated with wastewater treatment plant design when moving towards a more sustainable urban wastewater management. This integration allows an optimization of the design of both systems to achieve a better and more cost-effective wastewater management. Hitherto integrated process design has not been an option because the tools to predict in-sewer wastewater transformations have been inadequate. In this study the WATS model--being a new and validated tool for in-sewer microbial process simulations--is presented and its application for integrated sewer and treatment plant design is exemplified. A case study on a Malaysian catchment illustrates this integration. The effects of centralization of wastewater treatment and the subsequently longer transport distances are addressed. The layout of the intercepting sewer is optimized to meet the requirements of different treatment scenarios.

Half saturation constants for nitrate and nitrite by in-sewer anoxic transformations of wastewater organic matter.

A significant breakthrough and progress have been made in the study of the kinetics of microbial transformation in sewers under aerobic and under changing aerobic/anaerobic conditions. Fundamental knowledge on anoxic kinetics of wastewater is still lacking, so it is not now possible to apply an integrated approach to municipal wastewater treatment incorporating sewer networks as a bio-chemical reactor. This paper presents the results of studies on determining half saturation constants for nitrate, KNO3, and nitrite, KNO2, in raw wastewater. The average values of KNO3 and KNO2, determined from experiments conducted on 7 different wastewater samples were found to be 0.76 gNO3-N/m3 and 0.33 gNO2-N/m3 respectively.