A novel probe, based on vancomycin and 4-nitrobenzoxadiazole was synthesized and characterized, and used for the rapid and specific detection of Gram positive bacteria – the major pathogens responsible for eye infections in ocular specimens.
Abstract Background Slit2 is a ~ 200 kDa secreted glycoprotein that has been recently shown to regulate immune functions. However, not much is known about its role in HIV (human immunodeficiency virus)-1 pathogenesis. Results In the present study, we have shown that the N-terminal fragment of Slit2 (Slit2N) (~120 kDa) inhibits replication of both CXCR4 and CCR5-tropic HIV-1 viruses in T-cell lines and peripheral blood T-cells. Furthermore, we demonstrated inhibition of HIV-1 infection in resting CD4+ T-cells. In addition, we showed that Slit2N blocks cell-to-cell transmission of HIV-1. We have shown that Slit2N inhibits HIV-1 infection by blocking viral entry into T-cells. We also ruled out Slit2N-mediated inhibition of various other steps in the life cycle including binding, integration and viral transcription. Elucidation of the molecular mechanism revealed that Slit2N mediates its functional effects by binding to Robo1 receptor. Furthermore, we found that Slit2N inhibited Gp120-induced Robo1-actin association suggesting that Slit2N may inhibit cytoskeletal rearrangements facilitating HIV-1 entry. Studies into the mechanism of inhibition of HIV-1 revealed that Slit2N abrogated HIV-1 envelope-induced actin cytoskeletal dynamics in both T-cell lines and primary T-cells. We further showed that Slit2N specifically attenuated the HIV-1 envelope-induced signaling pathway consisting of Rac1, LIMK and cofilin that regulates actin polymerization. Conclusions Taken together, our results show that Slit2N inhibits HIV-1 replication through novel mechanisms involving modulation of cytoskeletal dynamics. Our study, thus, provides insights into the role of Slit2N in HIV-1 infection and underscores its potential in limiting viral replication in T-cells.
Background: Varicose veins are common vascular disorder. The study was conducted to analyse the clinical presentation, treatment options and complications of varicose veins.Methods: This observational study was conducted in Thanjavur Medical College Hospital. Adults with clinically diagnosed unilateral or bilateral varicose veins of lower limbs were studied in surgical wards between August 2013 to July 2014. The demographic data and presenting symptoms, signs and previous treatment were collected using a structured proforma. Thorough clinical examination, duplex scan and abdominal and pelvic examination were done in all cases to find out the secondary causes. Peripheral vascular system was examined. All the patients were followed regularly for the period of one month to one year after treatment.Results: A total of 60 cases were included. Most participants belonged to 2nd and 3rd decade of life. 55 (91%) participants were male and5 (9%) participants were females. 45 (75%) participants were agriculturists. 10 (17%) participants had bilateral involvement. Most participants 30 (50%) participants had 1 to 5 years duration of disease, most common clinical presentation of the study participants was varicosity with 70.0%, followed by lipodermo sclerosis, leg pain, hyperpigmentation, pruritus as 63.0%, 56.0%, 15% and 11.6% respectively. 17 (28.33%) patients were present with a venous ulcer. 32 (53%) participants were managed surgically, and 28 (47%) participants were managed conservatively. Most participants were treated with multiple ligation 21.8% followed by trendelenburg operation+ stripping18.75%.Conclusions: Regarding the treatment, surgery is the treatment of choice for primary varicose veins and conservative treatment for secondary varicose veins.
Tuberculosis continues to be a major public health concern, being the leading cause of death due to an infectious agent. Simultaneously, the prevalence of nontuberculous mycobacterial (NTM) infections worldwide has been rising [[1]Machado D. Couto I. Viveiros M. Advances in the molecular diagnosis of tuberculosis: from probes to genomes.Infect Genet Evol. 2019; 72: 93-112Crossref PubMed Scopus (15) Google Scholar]. Mycobacterium avium complex (MAC) is the most frequent NTM associated with pulmonary disease [[2]Somoskovi A. Mester J. Hale Y.M. Parsons L.M. Salfinger M. Laboratory diagnosis of nontuberculous mycobacteria.Clin Chest Med. 2002; 23: 585-597Summary Full Text Full Text PDF PubMed Scopus (49) Google Scholar]. MAC can often be misdiagnosed as Mycobacterium tuberculosis (Mtb), especially in countries with a high tuberculosis burden and treated with anti-tuberculosis drugs, to which MAC is usually not sensitive [[3]Pennington K.M. Vu A. Challener D. Rivera C.G. Shweta F.N. Zeuli J.D. Temesgen Z. Approach to the diagnosis and treatment of non-tuberculous mycobacterial disease.J Clin Tuberc Other Mycobact Dis. 2021; 24100244Crossref PubMed Scopus (4) Google Scholar]. Conventional laboratory diagnosis relies on culture, to see whether Mtb or NTM is present, and is hampered by the slow growth of most mycobacteria and may take several weeks. Several molecular tests such as the WHO-recommended Xpert MTB/RIF (Cepheid) assay have provided a rapid alternative to culture for detection of Mtb [[4]World Health OrganizationGlobal tuberculosis report 2013. World Health Organization, 2013Google Scholar]. However, most of these molecular Mtb assays do not detect NTM. In this article of EBioMedicine, Sarro and colleagues [[5]Sarro Y.D.S. et al.Development and clinical evaluation of a new multiplex PCR assay for a simultaneous diagnosis of tuberculous and nontuberculous mycobacteria.EBioMedicine. 2021; 70103527Summary Full Text Full Text PDF PubMed Scopus (3) Google Scholar] have developed a new highly sensitive multiplex MTB/NTM assay that can differentiate M. tuberculosis complex (MtbC) and the most common NTM, MAC. This multiplex assay has shown a high analytical sensitivity (5 CFUs/mL for TB and MAC and 20 CFUs/mL for other NTMs). The overall sensitivity, specificity, of the multiplex in cases without treatment failure were 83·3% and 96·6%, while the Xpert had value of 96·7% and 80·0%, respectively for the same patients compared to sputum culture. Thus, the method had a better specificity than Xpert for all TB-infection tested groups, although the Xpert had greater sensitivity. The assay also successfully detected all the MAC cases. Over the past two decades, molecular methods have come to represent a reliable and rapid alternative for laboratory diagnostics of mycobacteria in clinical samples. The most significant advance in recent times has been the FDA approval and global distribution of the Xpert MTB/RIF, which detects the Mtb complex as well as key genetic determinants of rifampin resistance. However, the main drawback of Xpert is that it is less sensitive than sputum culture and does not detect NTM infections. The most accurate current method for the detection and identification of NTM species is PCR-DNA sequencing with hsp65, rpoB, and 16S rRNA being the target genes [[6]Kim S.H. Shin J.H. Identification of nontuberculous mycobacteria using multilocous sequence analysis of 16S rRNA, hsp65, and rpoB.J Clin Lab Anal. 2018; 32: e22184Crossref Scopus (25) Google Scholar]. The advantage of PCR-sequencing is it is highly discriminatory, but the equipment and running costs are high. In contrast, real-time PCR is an alternative and cheaper technique. A few recent studies have evaluated real-time PCR for the detection of NTM [7Peixoto A.D. Montenegro L.M. Lima A.S. Melo F.L. Barbosa W.L. Neves M.M. et al.Identification of nontuberculous mycobacteria species by multiplex real-time PCR with high-resolution melting.Rev Soc Bras Med Trop. 2020; 53: e20200211Crossref PubMed Scopus (1) Google Scholar, 8Rocchetti T.T. Silbert S. Gostnell A. Kubasek C. Widen R. Validation of a multiplex real-time PCR assay for detection of mycobacterium spp., mycobacterium tuberculosis complex, and mycobacterium avium complex directly from clinical samples by use of the BD Max open system.J Clin Microbiol. 2016; 54: 1644-1647Crossref PubMed Scopus (14) Google Scholar, 9Sevilla I.A. Molina E. Elguezabal N. Pérez V. Garrido J.M. Juste R.A. Detection of mycobacteria, mycobacterium avium subspecies, and mycobacterium tuberculosis complex by a novel tetraplex real-time PCR assay.J Clin Microbiol. 2015; 53: 930-940Crossref PubMed Scopus (35) Google Scholar]. Peixoto et al. [[7]Peixoto A.D. Montenegro L.M. Lima A.S. Melo F.L. Barbosa W.L. Neves M.M. et al.Identification of nontuberculous mycobacteria species by multiplex real-time PCR with high-resolution melting.Rev Soc Bras Med Trop. 2020; 53: e20200211Crossref PubMed Scopus (1) Google Scholar] evaluated a multiplex real-time PCR with high-resolution melting for the identification and differentiation between Mtb and NTM, as well as among NTM species of clinical importance. However, this assay involved three different stages of PCR, one to differentiate Mtb and NTM and two more to differentiate the NTM species. Rocchetti et al. [[8]Rocchetti T.T. Silbert S. Gostnell A. Kubasek C. Widen R. Validation of a multiplex real-time PCR assay for detection of mycobacterium spp., mycobacterium tuberculosis complex, and mycobacterium avium complex directly from clinical samples by use of the BD Max open system.J Clin Microbiol. 2016; 54: 1644-1647Crossref PubMed Scopus (14) Google Scholar] used a multiplex real-time PCR on the BD Max open system to detect Mycobacterium spp. (pan-Mycobacterium), Mtb Complex group, and MAC group. Sevilla et al. [[9]Sevilla I.A. Molina E. Elguezabal N. Pérez V. Garrido J.M. Juste R.A. Detection of mycobacteria, mycobacterium avium subspecies, and mycobacterium tuberculosis complex by a novel tetraplex real-time PCR assay.J Clin Microbiol. 2015; 53: 930-940Crossref PubMed Scopus (35) Google Scholar] described the development and evaluation of a novel tetraplex real-time PCR for simultaneous detection of Mycobacterium genus, M. avium subspecies, and M. tuberculosis complex. Most recently, another duplex PCR targeting genes encoding catalase-peroxidase of MAC and Mtb to discriminate disseminated MAC and MtB infections in blood samples from HIV patients to diagnose disseminated tuberculosis was reported [[10]Sharma S. Latawa R. Wanchu A. Verma I. Differential diagnosis of disseminated mycobacterium avium and mycobacterium tuberculosis infection in HIV patients using duplex PCR.Future Microbiol. 2021; 16: 135-142Crossref PubMed Scopus (2) Google Scholar]. In this article, Sarro et al. have developed a novel one-step multiplex PCR platform that could be a convenient, cost-effective, and reliable method for simultaneous detection and differentiation between Mtb and MAC infections. This assay is extremely relevant to enhance discrimination of TB and NTM infections in pulmonary disease, with implications not only for selection of antimicrobial therapy diseases, but also for infection control. Though molecular methods have had a huge impact in the laboratory diagnosis of mycobacterial related infections and therefore patient care during the last decade, we need to be aware of their limitations. These include technical problems, expensive reagents, high labor costs, limited multiplexing capacity, technical skills requirement, and the requirement for specialized infrastructure. Furthermore, owing to their ubiquitous presence in the environment, a specific drawback in NTM diagnosis by PCR is that its detection in non-sterile samples does not equate infection. Hence, PCR assay tends to be a double-edged sword due to high sensitivity, making it challenging to discern NTM biological sample contamination from a true NTM infection. This assay is an open-system, usable on many PCR platforms but not yet automated to guarantee consistency in performance. Further development of this assay is needed, exploring the potential options for automation. Though culture presently continues to be the gold standard, being especially required for antibiotic susceptibility testing, the present one-step multiplex assay is a welcome addition to the promising pipeline of molecular diagnostics for diagnosis of mycobacterial infections. The assay has the potential to be adapted to a scalable, quick and easy point of care automated system and warrants further larger trials, particularly in areas where TB and various types of NTM disease are both prevalent. Appakkudal R Anand and Jyotirmay Biswas have no competing interests. Development and clinical evaluation of a new multiplex PCR assay for a simultaneous diagnosis of tuberculous and nontuberculous mycobacteriaOur new Multiplex assay demonstrates better specificity than Xpert for all group studied, in addition to detecting potential NTM cases. The assay could therefore complement the widely used Xpert assay and enhance discrimination of TB and NTM infections. Full-Text PDF Open Access
Abstract The secretory protein Slit2 and its receptors Robo1 and Robo4 are considered to regulate mobility and permeability of endothelial cells and other cell types. However, the roles of Slit2 and its two receptors in endothelial inflammatory responses remain to be clarified. In this study, we show that, in primary HUVECs, Slit2 represses LPS-induced secretion of certain inflammatory cytokines/chemokines, cell adhesion molecule ICAM-1 upregulation, and monocyte adhesion. Slit2’s anti-inflammatory effect is mediated by its dominant endothelial-specific receptor Robo4. However, the minor receptor Robo1 has proinflammatory properties and is downregulated by Slit2 via targeting of miR-218. Elucidation of molecular mechanism reveals that Slit2 represses inflammatory responses by inhibiting the Pyk2–NF-κB pathway downstream of LPS–TLR4. Further studies reveal that LPS enhances endothelial inflammation by downregulating the anti-inflammatory Slit2 and Robo4 in HUVECs in vitro, as well as in arterial endothelial cells and liver in vivo during endotoxemia. These results suggest that Slit2–Robo4 signaling is important in regulating LPS-induced endothelial inflammation, and LPS, in turn, enhances inflammation by interfering with the expression of the anti-inflammatory Slit2–Robo4 during the disease state. This implies that Slit2–Robo4 is a key regulator of endothelial inflammation, and its dysregulation during endotoxemia is a novel mechanism for LPS-induced vascular pathogenesis.
There is an error in Fig 3A, where the GAPDH results of Figs 1C and 2D were inadvertently included in Fig 3A during figure preparation.The updated Fig 3 provided with this notice shows the correct Fig 3A.The authors explain that the Figs 1C and 2D GAPDH results are identical as the results presented in these figures originate from the same experiment.The original data underlying the Fig 2A results are provided in S1 File below.
Background The pro-fibrogenic cytokine connective tissue growth factor (CTGF) plays an important role in the development and progression of fibrosis in many organ systems, including liver. However, its role in the pathogenesis of hepatitis C virus (HCV)-induced liver fibrosis remains unclear. Methods In the present study, we assessed CTGF expression in HCV-infected hepatocytes using replicon cells containing full-length HCV genotype 1 and the infectious HCV clone JFH1 (HCV genotype 2) by real-time PCR, Western blot analysis and confocal microscopy. We evaluated transforming growth factor β1 (TGF-β1) as a key upstream mediator of CTGF production using neutralizing antibodies and shRNAs. We also determined the signaling molecules involved in CTGF production using various immunological techniques. Results We demonstrated an enhanced expression of CTGF in two independent models of HCV infection. We also demonstrated that HCV induced CTGF expression in a TGF-β1-dependent manner. Further dissection of the molecular mechanisms revealed that CTGF production was mediated through sequential activation of MAPkinase and Smad-dependent pathways. Finally, to determine whether CTGF regulates fibrosis, we showed that shRNA-mediated knock-down of CTGF resulted in reduced expression of fibrotic markers in HCV replicon cells. Conclusion Our studies demonstrate a central role for CTGF expression in HCV-induced liver fibrosis and highlight the potential value of developing CTGF-based anti-fibrotic therapies to counter HCV-induced liver damage.
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