Month: October 2020

The ability to quantify macroscopic physiological parameters such as tissue contractile force and matrix elasticity cannot be achieved using current in vitro 2D cell culture systems. This study demonstrated that HCMV disrupts the mechanical force established by human fibroblasts within tissues and it is conceivable that the resulting tissue damage may contribute to the diverse pathologies associated with persistent HCMV infection in vivo. Several lines of evidence indicate that signaling triggered by CNP plays an important role in chondrocyte development. Upon CNP binding, its cognate receptor natriuretic peptide receptor 2 functions as a guanylyl cyclase to increase cyclic guanosine monophosphate levels in chondrocytes, female reproductive organs, and endothelial cells. Transgenic mice that overproduce CNP exhibit excessive growth, while defects of the CNP or Npr2 gene, leading to impairment of skeletal development. The increase in cGMP level activates cGMP-dependent protein kinase II and seems to promote the accumulation of extracellular matrix in the growth plate of CNPtransgenic mice. In human, overproduction of C-type natriuretic peptide due to a chromosomal translocation was reported to cause skeletal dysplasia associated with tall stature. In addition, acromesomelic dysplasia, type Maroteaux, ABT-199 characterized by dwarfism and short limbs, is caused by loss-offunction mutations in the Npr2 gene. On the other hand, NPR3, which is thought to act as a clearance receptor, knock-out mice resemble CNP transgenic mice. In this paper, we describe the first family with tall stature and macrodactyly of both great toes caused by a gain-of-function type mutation in the Npr2 gene. The mutant receptor, p.Val883Met, constitutively generates cGMP in vitro. Animal studies using the transgenic mice expressing the mutant NPR2 in chondrocytes demonstrated that skeletal overgrowth was associated with the overproduction of cGMP in cartilage. Our findings provide evidence that cGMP production downstream CNP/NPR2 system regulates the proliferation and differentiation of chondrocytes and determines skeletal growth. Here we have described a three-generation family with tall stature and macrodactyly due to a newly identified gain-offunction mutation of the Npr2 gene, p.Val883Met. Increased levels of cGMP both in blood samples of the patients and whole cell lysates expressing the mutant receptor clearly indicate its hypermorphism. Transgenic mice in which p.Val883Met mutant NPR2 was expressed in chondrocytes exhibited the elevated cGMP concentration and the excessive growth and deformities of vertebrae and long bones, which reproduced the symptoms observed in the patients. Taken together, our data are compatible with the interpretation that p.Val883Met mutant is constitutively active and causes skeletal overgrowth by increasing the level of cGMP in chondrocytes. However, we cannot exclude the possibility that the high expression level of NPR2 also contributed to overgrowth in transgenic mice to some extent. Histological examination confirmed that the skeletal overgrowth was caused by the widening of the growth plates in the transgenic mice expressing the mutant Npr2. As expected, the histological finding was similar to, but severer than, that reported in the mice with overexpression of CNP in chondrocytes. The disorganized arrangement of chondrocytes in the transgenic mice indicates the critical role of the CNP/NPR2 pathway in chondrogenesis.

Mitochondrial permeability transition and calcium uptake were increased in DKO cardiomyocytes and mitochondrial respiration rate using skinned fibers from DKO myocardium were BYL719 in vivo reduced compared with wild type. Therefore, beyond the DKO model affecting both CRYAB and HSPB2 expression, a new mouse model that targets specifically hspb2 is required to determine the distinct tissue-specific functions of HSPB2 in vivo. The present study reports the creation of a conditional floxed hspb2 allele and the production of mice with a cardiac-specific knockout of hspb2. Our data reveal that the absence of HSPB2 in the heart does not significantly affect the cardiac hypertrophic response to pressure overload stimuli, but that HSPB2 deficiency depresses mitochondrial fatty acid betaoxidation and ATP production under these conditions. In this study, we describe a new mouse model characterized by the cardiac specific deletion of the hspb2 gene. Under normal conditions, HSPB2cKO did not exhibit any obvious cardiac anomaly and this is consistent with the observations made with the DKO animals, which were deficient in both hspb2 and hspb5. We further demonstrated that lack of HSPB2 did not modify the cardiac response to pressure overload in response to either mild or severe stress. Because the expression levels for some other sHSPs such as HSPB1, HSPB5 and HSPB6 were not visibly altered, absence of phenotype in HSPB2cKO does not seem to be linked to major compensatory responses. As the present study analyzes a cardiac-specific knockout of hspb2, we could not definitively address the role of HSPB2 deficiency in other organs especially in skeletal muscles that harbor high levels of HSPB2 expression. The superfamily of small MW HSPs has been implicated in diverse functions and biological roles ranging from cellular immunity to oncogenesis to cardiomyopathy and heart failure. Whereas both cryab and hspb2 are arranged adjacently in the genome, we have hypothesized the existence of tissue-specific functions for their expression, under the control of myogenic regulators that drive high levels of endogenous expression in skeletal muscle and the heart. Unlike distinct mutations in human CryAB that have been linked to various inherited multisystem diseases, HSPB2 is not only the most divergent sHSP family but its biological function remains unknown. We confirm that HSPB2, like CryAB, is dispensable for cardiac function and maintenance of myocardial integrity. Similarly the knockout of hspb1, another sHSP highly expressed during heart development did not provoke any major disturbance in cardiac anatomy or function as evidenced by the normal lifespan of those animals. Although prior studies by other investigators have shown that isolated and intact hearts lacking both CRYAB and HSPB2 exhibit severe contractile dysfunction and increased myocardial injury in response to ischemia/reperfusion ex vivo, our laboratory has reported increased resistance of DKO hearts to in situ and ex vivo ischemic conditions compared with wild-type controls. Such studies, therefore, have provided confounding insights about the functional roles of these two sHSPs in ischemic cardioprotection and in no study could the specific role of HSPB2, in particular, be unambiguously assigned. In addition, we found that HSPB2 appears to be required for systolic performance and for maintaining cardiac energetics in the isolated perfused mouse heart. To address these unmet needs, we have advocated the use of genetic tools to unmask potential novel and non-redundant functions between CryAB and HSPB2 in terms of cardiac mechanics and energetics.

If the Kras coding sequence is replaced by Hras, the embryonic lethality is rescued but adult animals develop dilated cardiomyopathy and elevated blood pressure, suggesting a unique role of KRAS in cardiovascular homeostasis. Homozygous Nras KO mice are overall Pazopanib healthy, but present impaired antiviral immune response and T-cell function due to a reduced population of CD8+ cells in the thymus. After influenza virus infection, Nras KO mice showed a reduced response of CD4+ T lymphocytes, granulocytes, NK cells, macrophages, and CD8+ T lymphocytes. Notably the overall levels of RAS proteins in Nras KO mice remain unchanged due to a compensatory increase of KRAS and HRAS, suggesting a specific role for NRAS in lymphoid cells. The role of elevated RAS in cancer has been investigated in several mouse models. Myeloid malignancies with incomplete penetrance and long latency periods were observed when the bone marrow of irradiated mice was repopulated with cells overexpressing a constitutively active NRAS protein. Several animals presented increased numbers of granulocytes at the expense of lymphocytes, but due to the long latency and low penetrance, it was suggested that a secondary hit is required to induce cancer. However, when NRASG12D was expressed in early hematopoietic cells from an MSCV retroviral vector, myeloid disorders resembling human AML and CMML were efficiently induced by higher and lower NRAS signaling levels respectively. Heterozygous expression of NRASG12D fromthe endogenousNras locus in epiblast cells results in embryonic lethality. Heterozygous expression onlyinliver, spleenand bonemarrow resultsinamild phenotype dependent on genetic background and characterized by a wide spectrum of hematologic diseases. However, upon infection with the MOL4070LTR retrovirus, these animals developed AML considerably faster than control mice. Homozygous expression leads to myeloid hyperplasia with shorter latency. In semisolid culture, the bone marrow cells displayed an abnormal growth pattern, and after co-transplantation with competitor wild type cells into irradiated recipient mice, a dose-dependent phenotype was observed. Almost all animals with heterozygous expression of NrasG12D developed a CMML-like disease starting after 6 months. The mice suffered from anemia and developed myeloid hyperplasia in bone marrow and spleen. Homozygous expression did not induce sustained CMML but resulted in acute T-cell lymphoblastic leukemia/lymphoma. Increment of the number of transplanted cells changed the disease pattern of the recipient mice, as lethal myeloproliferative disease was induced in the majority of the mice independent on the number of NrasG12D alleles. Incidences resembling human B- and T-cell lymphoblastic leukemia/lymphoma were likewise observed but only upon transplantation of NrasG12D/G12D cells. Despitethe examples of overexpression ofwtNrasin human cancer and the large number of disease mouse models with deregulated Nras expression, onlytransgenic mice overexpressing wildtypeNras under the mouse mammary tumor virus long terminal repeat are earlier described. These developed hyperplasias and malignant tumors in the tissues with the highest transgene expression. We have recently developed a set of mouse models in which the murine leukemia virus Akv 1–99 long terminal repeat was introduced within the Nras locus at the exact positions of previously identified retroviralintegrations. Here we report on the early post natal pathology which is the consequence of an upregulated wt Nras expression from its endogenous locus, which is particularly increased in spleen.

This situation may be reversed following treatment; in fact, it has been reported that anti-TNF therapy in RA is associated with an increased percentage of circulating Th17 cells possibly attributable to a decreased homing to the synovium. We were particularly interested in examining IL-17 production by other cell populations, such as cd T cells, CD8 T cells and NK cells, but observed that this cytokine was exclusively produced by CD4+ T cells in RA synovial fluid and in the peripheral blood of healthy controls, eRA and established RA patients, consistent with work published by Shen et al. Th17 biology is complex and incompletely understood, which may explain why previous reports on the frequency of Th17 cells in RA have yielded conflicting results. Some authors have found increased circulating Th17 frequencies whereas others report comparable frequencies to those displayed by healthy controls. Discordances among studies may be due in part to differences in the clinical profile and/or medications of patients. Whereas an influence of immunomodulating drugs on the ex vivo behavior of isolated cells is possible, the effect of perpetuating inflammatory feedback loops that are effective as disease progresses may also be a player. Our population of subjects with early disease and who had never received corticosteroids or DMARDs was not homogeneous when considering the frequency of circulating Th17 cells. A clear difference between anti-CCP+ and anti-CCP- eRA patients was observed, and a ready explanation for this observation is not apparent at present. In fact, the data reported herein may contribute to reinforce the notion that anti-CCP+ and anti-CCPRA are distinct entities. The different circulating Th17 and Th17/Th1 cell frequencies in patients with early versus established RA suggests that the immune mechanisms mediating early phases of the disease may be different from those implicated in more advanced stages. Interestingly, when our anti-CCP+ eRA patients were reexamined one year after the first visit, while receiving treatment with oral MTX, their circulating Th17 frequency was no longer different from controls. This was apparent not only in patients who had achieved remission but also in patients who had persistent disease activity, which suggests that medication may be playing a role. Other investigators have concluded that the peripheral blood Th17 frequency in RA may be modified by therapy. As noted above, Aerts et al reported that in established RA the peripheral Th17 cell frequency is not elevated, but anti-TNF therapy induces a striking increase of circulating Th17 cells and IL-17 production, irrespective of disease activity. In addition, Notley et al found that in Reversine collagen-induced arthritis, TNF blockade resulted in reduced arthritis severity but, unexpectedly, expanded populations of extra-articular Th17 cells, which were shown by adoptive transfer to be pathogenic. Finally, recently published phase II clinical trials on the efficacy of IL-17 blockade in RA have yielded conflicting results. In light of our findings, discrepancies could be related to differences in the stage of disease or anti-CCP antibody status of patients. Breast cancer is one of the most frequently diagnosed cancers in women and is the leading cause of cancer-related death among women worldwide, including China. In the United States, it has been estimated that approximately 12% of women will develop breast cancer at some point in their lives. The vast majority of breast cancer-related deaths are due to metastasis, and the lung, liver, bone and brain are the most prevalent sites. Metastasis itself is a well-orchestrated multi-step process. It includes tumor cell invasion of the basement membrane.

Genomic, DNA-based molecular methods of species identification are advantageous as they can be applied to specimens and situations unsuitable for morphological taxonomy. However, PCR-based molecular identification methods require relatively expensive and sophisticated laboratory equipment unavailable to public health practitioners in many developing countries. Recently, loop mediated isothermal amplification technology was adapted for molecular discrimination between An. gambiae and An. arabiensis mosquito species, showing a sensitivity better than 0.9 and a 100% specificity compared with standard rDNA-PCR when testing field-captured mosquitoes. The use of isothermal amplification dramatically simplifies the molecular identification Fulvestrant process, greatly simplifies the instrumentation needed, and even allows instrument-free operation. In recent years, there have been considerable efforts to integrate biochemical analysis and medical diagnostics processes into monolithic microfluidic platforms. Compared to conventional laboratory methods, such integrated microfluidic implementations offer the advantages of low cost, short test times, small sample sizes, low reagent consumption, and most importantly, full automation of all processes from sample preparation to detection in a single device. The “sample-to-answer” capability is particularly attractive for resource-poor regions, where funds and trained personnel are in short supply. Although a number of groups are developing microfluidic components for nucleic acid testing, there are, to date, only a few reports of fully-integrated, microfluidic NAT chips that can perform all the necessary steps from sample introduction and preparation to target detection. On-chip sample preparation is still a challenge. In an effort to simplify the design and operation of a microfluidic diagnostics system, our group has recently developed a multi-function, isothermal amplification reactor with an embedded isolation membrane, such as Flinders Technology Associates Whatman FTAH, without a need for nucleic acid elution. The porous cellulose FTA membrane serves as a solid-phase binding medium for extraction, concentration, and purification of nucleic acids from cell lysates. We have used our devices to detect the presence of the HIV virus in saliva specimens. Our chip with an embedded FTA membrane successfully isolated viral RNA and carried out real-time, reverse-transcription, loopmediated isothermal amplification with a detection limit better than ten target particles per sample. In this paper, we will show that a similar idea can be used to distinguish mosquito species. Briefly, we report on a simple, lowcost, disposable, sample-to-answer, microfluidic chip, which integrates the functional steps of lysis of mosquito tissue cells; nucleic acid capture, concentration, and purification; isothermal amplification; and detection into a single chamber formed in a plastic substrate. A cell phone with an embedded CCD camera monitors the fluorescence signal emitted during the enzymatic amplification reaction in the microfluidic chip. We use a small piece of Whatman FTA filter paper for sample collection and nucleic acid isolation. The utility of our system was demonstrated by identifying the malaria-transmitting mosquitoes An. gambiae and An. arabiensis. To the best of our knowledge, this is the first report describing an integrated microfluidic chip for molecular identification of insect disease vectors with a cell phone recorder. The chip design and operation, including cell phone imaging for detection, can readily accommodate multiplexed analysis for parallel detection of several mosquito species and appropriate control reactions.