Diagnosis of abdominal tuberculosis: Detection of mycobacterial CFP-10 and HspX proteins by gold nanoparticle-PCR amplified immunoassay.

Attempts were made to improve the efficacy of PCR amplified immunoassay (I-PCR) for diagnosing abdominal TB cases by utilizing the gold nanoparticle (AuNP)-based I-PCR, where AuNPs were functionalized with detection antibodies/oligonucleotides that exhibited 84.3% sensitivity and 95.1% specificity. This assay would improve the ongoing algorithms used in abdominal TB diagnosis.

Variable antiviral activity of islatravir against M184I/V mutant HIV-1 selected during antiretroviral therapy.

BACKGROUND: Islatravir is a new antiretroviral drug that inhibits the reverse transcriptase (RT) of HIV-1 through multiple mechanisms. It is proposed to be used in combination with doravirine, a new NNRTI. M184V/I mutations have been shown to reduce the in vitro antiviral activity of islatravir, but their effect when pre-selected during ART has not been investigated. METHODS: HIV-1 rt sequences were obtained from four individuals of the Garrahan HIV cohort prior to, or during virological failure to ART. HIV-1 infectious molecular clones were constructed on an NL4-3 backbone, and infectious viruses were produced by transfection of 293T cells. Fold-changes in IC50 were calculated for each mutant versus the NL4-3 WT. HIV-1 phenotypic drug resistance was tested in vitro against NRTIs and NNRTIs. RESULTS: In all the cases, M184I/V, either alone or in the presence of other mutations, was associated with reduced susceptibility to islatravir, abacavir and lamivudine. Viruses carrying M184V/I showed variable levels of resistance to islatravir (4.8 to 33.8-fold). The greatest reduction in susceptibility was observed for viruses carrying the mutations M184V + V106I (33.8-fold resistance) or M184V + I142V (25.2-fold resistance). For NNRTIs, the presence of V106I alone did not affect susceptibility to doravirine or etravirine, but showed a modest reduction in susceptibility to efavirenz (6-fold). Susceptibility to doravirine was slightly reduced only for one of the mutants carrying V106I in combination with Y181C and M184V. CONCLUSIONS: Mutations and polymorphisms selected in vivo together with M184V/I depend on the viral genetic context and on ART history, and could affect the efficacy of islatravir once available for use in the clinic.

Antiviral potency of long-acting islatravir subdermal implant in SHIV-infected macaques.

Treatment nonadherence is a pressing issue in people living with HIV (PLWH), as they require lifelong therapy to maintain viral suppression. Poor adherence leads to antiretroviral (ARV) resistance, transmission to others, AIDS progression, and increased morbidity and mortality. Long-acting (LA) ARV therapy is a promising strategy to combat the clinical drawback of user-dependent dosing. Islatravir (ISL) is a promising candidate for HIV treatment given its long half-life and high potency. Here we show constant ISL release from a subdermal LA nanofluidic implant achieves viral load reduction in SHIV-infected macaques. Specifically, a mean delivery dosage of 0.21 ± 0.07 mg/kg/day yielded a mean viral load reduction of -2.30 ± 0.53 log10 copies/mL at week 2, compared to baseline. The antiviral potency of the ISL delivered from the nanofluidic implant was higher than oral ISL dosed either daily or weekly. At week 3, viral resistance to ISL emerged in 2 out of 8 macaques, attributable to M184V mutation, supporting the need of combining ISL with other ARV for HIV treatment. The ISL implant produced moderate reactivity in the surrounding tissue, indicating tolerability. Overall, we present the ISL subdermal implant as a promising approach for LA ARV treatment in PLWH.

Effect of Succinamic Acid Derivative on the Growth Kinetics and Induction of Apoptosis in a Cancer Cell Line.

Introduction Head and neck cancers are heterogeneous malignancies associated with significant morbidity. Oral cancers are related to the use of tobacco products. Smokeless tobacco usage is a health problem worldwide, and its carcinogenic mechanism is largely unknown. Despite advances in conventional treatments, side effects and drug resistance remain unsolved. Therefore, novel therapeutic agents with minimal side effects using plant derivatives should be explored. An active antihyperglycemic and antioxidant compound known as FIIc was isolated from the fruit pulp of Eugenia jambolana (US Patent No.: 2,30,753). Although E. jambolana is reported to have anticancer activity, no study has been reported on its growth kinetics and apoptotic potential in the human head and neck cancer cell line (SCC4). The present study evaluated the effect of an herbal compound isolated from the fruit pulp of E. jambolana and chemically synthesized the same compound, α-hydroxy succinamic acid (α-HSA), on SCC4 proliferation and apoptotic gene expression. Methods The SCC4 cell line was cultured in Dulbecco's Modified Eagle Medium (DMEM). The dosages of smokeless tobacco extract (STE), herbal compound, and synthetic compound were determined by cell viability assay, and their effect on mRNA expression of apoptotic genes was measured by real-time polymerase chain reaction. Results The present study observed significant therapeutic effects of the natural and synthetic compounds from the fruit pulp of E. jambolana at the concentration range of 100-200 µg/mL on the SCC4 cell line. α-HSA had antiproliferative action; upregulated apoptotic genes like p53, p21, and Bax; and downregulated anti-apoptotic genes like survivin in the SCC4 cell line. Conclusion The therapeutic potential of α-HSA and the putative mechanisms involved may be explored to provide the basis for future therapeutic interventions in oral cancer mediated by smokeless tobacco.

A Recombinant Protein XBB.1.5 RBD/Alum/CpG Vaccine Elicits High Neutralizing Antibody Titers against Omicron Subvariants of SARS-CoV-2.

(1) Background: We previously reported the development of a recombinant protein SARS-CoV-2 vaccine, consisting of the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein, adjuvanted with aluminum hydroxide (alum) and CpG oligonucleotides. In mice and non-human primates, our wild-type (WT) RBD vaccine induced high neutralizing antibody titers against the WT isolate of the virus, and, with partners in India and Indonesia, it was later developed into two closely resembling human vaccines, Corbevax and Indovac. Here, we describe the development and characterization of a next-generation vaccine adapted to the recently emerging XBB variants of SARS-CoV-2. (2) Methods: We conducted preclinical studies in mice using a novel yeast-produced SARS-CoV-2 XBB.1.5 RBD subunit vaccine candidate formulated with alum and CpG. We examined the neutralization profile of sera obtained from mice vaccinated twice intramuscularly at a 21-day interval with the XBB.1.5-based RBD vaccine, against WT, Beta, Delta, BA.4, BQ.1.1, BA.2.75.2, XBB.1.16, XBB.1.5, and EG.5.1 SARS-CoV-2 pseudoviruses. (3) Results: The XBB.1.5 RBD/CpG/alum vaccine elicited a robust antibody response in mice. Furthermore, the serum from vaccinated mice demonstrated potent neutralization against the XBB.1.5 pseudovirus as well as several other Omicron pseudoviruses. However, regardless of the high antibody cross-reactivity with ELISA, the anti-XBB.1.5 RBD antigen serum showed low neutralizing titers against the WT and Delta virus variants. (4) Conclusions: Whereas we observed modest cross-neutralization against Omicron subvariants with the sera from mice vaccinated with the WT RBD/CpG/Alum vaccine or with the BA.4/5-based vaccine, the sera raised against the XBB.1.5 RBD showed robust cross-neutralization. These findings underscore the imminent opportunity for an updated vaccine formulation utilizing the XBB.1.5 RBD antigen.

Engineered CD4 T cells expressing a membrane anchored viral inhibitor restrict HIV-1 through cis and trans mechanisms.

HIV-1 infection of target cells can occur through either cell-free virions or cell-cell transmission in a virological synapse, with the latter mechanism of infection reported to be 100- to 1,000-fold more efficient. Neutralizing antibodies and entry inhibitors effectively block cell-free HIV-1, but with few exceptions, they display much less inhibitory activity against cell-mediated HIV-1 transmission. Previously, we showed that engineering HIV-1 target cells by genetically linking single-chain variable fragments (scFvs) of antibodies to glycosyl phosphatidylinositol (GPI) potently blocks infection by cell-free virions and cell-mediated infection by immature dendritic cell (iDC)-captured HIV-1. Expression of scFvs on CD4+ cell lines by transduction with X5 derived anti-HIV-1 Env antibody linked to a GPI attachment signal directs GPI-anchored scFvs into lipid rafts of the plasma membrane. In this study, we further characterize the effect of GPI-scFv X5 on cell-cell HIV-1 transmission from DCs to target cells. We report that expression of GPI-scFv X5 in transduced CD4+ cell lines and human primary CD4+ T cells potently restricts viral replication in iDC- or mDC-captured HIV-1 in trans. Using live-cell imaging, we observed that when GPI-GFP or GPI-scFv X5 transduced T cells are co-cultured with iDCs, GPI-anchored proteins enrich in contact zones and subsequently migrate from T cells into DCs, suggesting that transferred GPI-scFv X5 interferes with HIV-1 infection of iDCs. We conclude that GPI-scFv X5 on the surface of transduced CD4+ T cells not only potently blocks cell-mediated infection by DCs, but it transfers from transduced cells to the surface of iDCs and neutralizes HIV-1 replication in iDCs. Our findings have important implications for HIV-1 antibody-based immunotherapies as they demonstrate a viral inhibitory effect that extends beyond the transduced CD4+ T cells to iDCs which can enhance HIV-1 replication.

Quantification of mycobacterial proteins in extrapulmonary tuberculosis cases by nano-based real-time immuno-PCR.

Aim: Diagnosis of extrapulmonary tuberculosis (EPTB) is difficult, and a rapid and dependable diagnostic test is urgently needed. Methods: A nano-based assay, SYBR Green magnetic bead-coupled gold nanoparticle-based real-time immuno-polymerase chain reaction (MB-AuNP-RT-I-PCR) was studied for the quantitative detection of Mycobacterium tuberculosis MPT-64+CFP-10 proteins in clinically suspected EPTB patients. Results: A wide range (270 fg/ml-9.9 ng/ml) of MPT-64+CFP-10 was quantified by MB-AuNP-RT-I-PCR in EPTB cases, whereas magneto-ELISA demonstrated a narrow range (1.8-10 ng/ml). Furthermore, high sensitivity (88.2%) and specificity (100%) were attained by MB-AuNP-RT-I-PCR in EPTB (n = 51) and non-TB control (n = 49) subjects, respectively. Both MB-AuNP-I-PCR/magneto-ELISA exhibited significantly lower (p < 0.05-0.01) sensitivities than MB-AuNP-RT-I-PCR. Conclusion: The MB-AuNP-RT-I-PCR described herein shows good diagnostic accuracy, which may translate into a credible diagnostic kit.

Extrapulmonary tuberculosis (EPTB) is a type of tuberculosis disease caused by the bacteria Mycobacterium tuberculosis (Mtb) that affect other regions of the body, rather than the lungs. Detecting EPTB is difficult, and a fast and reliable test is needed. This study developed a test based on a small particle, known as a nanoparticle, to identify Mtb in people with EPTB. The test shows good accuracy and could be used for routine testing.

Diagnosis of genitourinary tuberculosis: detection of mycobacterial lipoarabinomannan and MPT-64 biomarkers within urine extracellular vesicles by nano-based immuno-PCR assay.

We detected a cocktail of Mycobacterium tuberculosis lipoarabinomannan (LAM) and MPT-64 biomarkers within urine extracellular vesicles (EVs) of genitourinary TB (GUTB) patients by nano-based immuno-PCR (I-PCR) assay, i.e., magnetic bead-coupled gold nanoparticle-based I-PCR (MB-AuNP-I-PCR) and compared the results with I-PCR and Magneto-ELISA. The size (s) of urine EVs ranged between 52.6 and 220.4 nm as analyzed by transmission electron microscopy (TEM) and nanoparticle tracking analysis. Functionalized AuNPs (coupled with detection antibodies/oligonucleotides) were characterized by UV-vis spectroscopy, TEM, ELISA, PCR, Atomic Force Microscopy and Fourier Transform Infrared spectroscopy, while conjugation of capture antibodies with MBs was validated by UV-vis spectroscopy and Magneto-ELISA. Our MB-AuNP-I-PCR exhibited sensitivities of 85% and 87.2% in clinically suspected (n = 40) and total (n = 47) GUTB cases, respectively, with 97.1% specificity in non-TB controls (n = 35). These results were further authenticated by the quantitative SYBR Green MB-AuNP-real-time I-PCR (MB-AuNP-RT-I-PCR). Concurrently, I-PCR and Magneto-ELISA showed sensitivities of 68.1% and 61.7%, respectively in total GUTB cases, which were significantly lower (p < 0.05-0.01) than MB-AuNP-I-PCR. Markedly, a wide range (400 fg/mL-11 ng/mL) of LAM+MPT-64 was quantified within urine EVs of GUTB cases by SYBR Green MB-AuNP-RT-I-PCR, which can assess the disease dynamics. This study will certainly improve the current algorithms used in GUTB diagnostics.

Modulation of glucose metabolism-related genes in diabetic rats treated with herbal synthetic anti-diabetic compound (α-HSA): insights from transcriptomic profiling.

OBJECTIVES: Eugenia jambolana is a medicinal plant traditionally used for treating diabetes. The bioactive compound FIIc, which is derived from the fruit pulp of E. jambolana, has been identified and purified as α-HSA. Previous studies have demonstrated that administration of α-HSA for 6 weeks improved glycemic index and dyslipidemia in rats with T2D. This study investigated the molecular mechanism underlying the potential therapeutic effects of α-HSA in experimentally induced diabetic rats. METHODS: Male Wistar rats were divided into four groups: diabetic control, diabetic treated with FIIc, diabetic treated with α-HSA, and diabetic treated with glibenclamide. Over a 6-week experimental period, transcriptomic analysis was conducted on liver, skeletal, and pancreatic tissue samples collected from the rats. RESULTS: The study findings revealed significant upregulation of genes associated with glucose metabolism and insulin signaling in the groups treated with FIIc and α-HSA, compared to the diabetic control group. Moreover, pro-inflammatory genes were downregulated in these treatment groups. These results indicate that α-HSA has the potential to modulate key metabolic pathways, improve glucose homeostasis, enhance insulin sensitivity, and alleviate inflammation. CONCLUSIONS: This study provides compelling scientific evidence supporting the potential of α-HSA as a therapeutic agent for diabetes treatment. The observed upregulation of genes related to glucose metabolism and insulin signaling, along with the downregulation of pro-inflammatory genes, aligns with the pharmacological activity of α-HSA in controlling glucose homeostasis and improving insulin sensitivity. These findings suggest that α-HSA holds promise as a novel therapeutic approach for managing diabetes and its associated complications.

Genetic Mechanisms for Hybrid Breeding in Vegetable Crops.

To address the complex challenges faced by our planet such as rapidly changing climate patterns, food and nutritional insecurities, and the escalating world population, the development of hybrid vegetable crops is imperative. Vegetable hybrids could effectively mitigate the above-mentioned fundamental challenges in numerous countries. Utilizing genetic mechanisms to create hybrids not only reduces costs but also holds significant practical implications, particularly in streamlining hybrid seed production. These mechanisms encompass self-incompatibility (SI), male sterility, and gynoecism. The present comprehensive review is primarily focused on the elucidation of fundamental processes associated with floral characteristics, the genetic regulation of floral traits, pollen biology, and development. Specific attention is given to the mechanisms for masculinizing and feminizing cucurbits to facilitate hybrid seed production as well as the hybridization approaches used in the biofortification of vegetable crops. Furthermore, this review provides valuable insights into recent biotechnological advancements and their future utilization for developing the genetic systems of major vegetable crops.

Long-acting refillable nanofluidic implant confers protection against SHIV infection in nonhuman primates.

The impact of pre-exposure prophylaxis (PrEP) on slowing the global HIV epidemic hinges on effective drugs and delivery platforms. Oral drug regimens are the pillar of HIV PrEP, but variable adherence has spurred development of long-acting delivery systems with the aim of increasing PrEP access, uptake, and persistence. We have developed a long-acting subcutaneous nanofluidic implant that can be refilled transcutaneously for sustained release of the HIV drug islatravir, a nucleoside reverse transcriptase translocation inhibitor that is used for HIV PrEP. In rhesus macaques, the islatravir-eluting implants achieved constant concentrations of islatravir in plasma (median 3.14 nM) and islatravir triphosphate in peripheral blood mononuclear cells (median 0.16 picomole per 106 cells) for more than 20 months. These drug concentrations were above the established PrEP protection threshold. In two unblinded, placebo-controlled studies, islatravir-eluting implants conferred 100% protection against infection with SHIVSF162P3 after repeated low-dose rectal or vaginal challenge in male or female rhesus macaques, respectively, compared to placebo control groups. The islatravir-eluting implants were well tolerated with mild local tissue inflammation and no signs of systemic toxicity over the 20-month study period. This refillable islatravir-eluting implant has potential as a long-acting drug delivery system for HIV PrEP.

Psychiatric comorbidities among people with epilepsy: A population-based assessment in disadvantaged communities.

Psychiatric disorders are frequent among people with epilepsy but often under-recognized. The diagnosis and treatment of these disorders in low- and low-middle-income countries (LMICs) are challenging. METHODS: This cross-sectional survey included people recruited during a community epilepsy screening program involving 59,509 individuals from poor communities in Ludhiana in Northwest India. Adults (age ≥18 years) with confirmed epilepsy on antiseizure medications were screened for depression and anxiety using the Neurological Disorders Depression Inventory for Epilepsy (NDDI-E) and Generalized Anxiety Disorder-7 (GAD-7) twice over two years of follow-up. They were later interviewed for symptoms using the Brief Psychiatric Rating Scale, which was then confirmed by assessments by an experienced psychiatrist. RESULTS: Of the 240 people with confirmed epilepsy, 167 (70%) were adults, of whom, 116 (70%) eventually participated in the study. The NDDI-E with a cut-off of 15 identified depression in 14 (12%) of 116 people after one year of follow-up and 17 (15%) at two years. The GAD-7 using a cut-off of 6 identified 22 (19%) at one year and 32 (28%) with anxiety at two years. The area under the curves for NDDI-E was estimated as 0.62 (95%CI, 0.51-0.73; SE: 0.06; p = 0.04) and for GAD-7 as 0.62 (95%CI, 0.46-0.78; SE: 0.08; p = 0.12). Brief Psychiatric Rating Scale identified 63 (54%) people with psychiatric symptoms, for whom, a psychiatric diagnosis was confirmed in 60 (52%). A psychiatric diagnosis was associated with education below high school [Odds Ratio (OR): 2.59, 95%CI, 1.12-5.1; p = 0.03], later age of seizure onset (OR, 1.05, 95%CI: 1.0-1.10; p = 0.04), seizure frequency of at least one/year at enrolment (OR, 2.36, 95%CI: 1.0-5.58; p = 0.05) and the use of clobazam (OR, 5.09, 95%CI, 1.40-18.42; p = 0.01). CONCLUSION: Depression and anxiety are common in people with epilepsy. Our findings underscore the low yields of screening instruments, NDDI-E and GAD-7, and comparatively better professionally-administered diagnostic assessments in resource-limited settings in LMICs. Moreover, previously established cut-offs do not apply to the community studied.

A cluster-randomized trial comparing home-based primary health care and usual clinic care for epilepsy in a resource-limited country.

OBJECTIVE: To ascertain whether home-based care with community and primary healthcare workers' support improves adherence to antiseizure medications, seizure control, and quality of life over routine clinic-based care in community samples of people with epilepsy in a resource-poor country. METHODS: Participants included consenting individuals with active epilepsy identified in a population survey in impoverished communities. The intervention included antiseizure medication provision, adherence reinforcement and epilepsy self- and stigma management guidance provided by a primary health care-equivalent worker. We compared the intervention group to a routine clinic-based care group in a cluster-randomized trial lasting 24 months. The primary outcome was antiseizure medication adherence, appraised from monthly pill counts. Seizure outcomes were assessed by monthly seizure aggregates and time to first seizure and impact by the Personal Impact of Epilepsy scale. RESULTS: Enrolment began on September 25, 2017 and was complete by July 24, 2018. Twenty-four clusters, each comprising ten people with epilepsy, were randomized to either home- or clinic-care. Home-care recipients were more likely to have used up their monthly-dispensed epilepsy medicine stock (regression coefficient: 0.585; 95% confidence intervals, 0.289-0.881; P = 0.001) and had fewer seizures (regression coefficient: -2.060; 95%CI, -3.335 to -0.785; P = 0.002). More people from clinic-care (n = 44; 37%) than home-care (n = 23; 19%) exited the trial (P = 0.003). The time to first seizure, adverse effects and the personal impact of epilepsy were similar in the two arms. SIGNIFICANCE: Home care for epilepsy compared to clinic care in resource-limited communities improves medication adherence and seizure outcomes and reduces the secondary epilepsy treatment gap.

Genomic landscape of the DHA1 family in Candida auris and mapping substrate repertoire of CauMdr1.

The last decade has witnessed the rise of an extremely threatening healthcare-associated multidrug-resistant non-albicans Candida (NAC) species, Candida auris. Since besides target alterations, efflux mechanisms contribute maximally to antifungal resistance, it is imperative to investigate their contributions in this pathogen. Of note, within the major facilitator superfamily (MFS) of efflux pumps, drug/H+ antiporter family 1 (DHA1) has been established as a predominant contributor towards xenobiotic efflux. Our study provides a complete landscape of DHA1 transporters encoded in the genome of C. auris. This study identifies 14 DHA1 transporters encoded in the genome of the pathogen. We also construct deletion and heterologous overexpression strains for the most important DHA1 drug transporter, viz., CauMdr1 to map the spectrum of its substrates. While the knockout strain did not show any significant changes in the resistance patterns against most of the tested substrates, the ortholog when overexpressed in a minimal background Saccharomyces cerevisiae strain, AD1-8u-, showed significant enhancement in the minimum inhibitory concentrations (MICs) against a large panel of antifungal molecules. Altogether, the present study provides a comprehensive template for investigating the role of DHA1 members of C. auris in antifungal resistance mechanisms. KEY POINTS: • Fourteen putative DHA1 transporters are encoded in the Candida auris genome. • Deletion of the CauMDR1 gene does not lead to major changes in drug resistance. • CauMdr1 recognizes and effluxes numerous xenobiotics, including prominent azoles.

Quantitative detection of mycobacterial mannophosphoinositides in tuberculosis patients by real-time immuno-PCR assay.

A real-time immuno-PCR assay was deliberated to detect mycobacterial mannophosphoinositides (PIMs). A dynamic range of PIMs (0.9 pg/mL-10 ng/mL) was detected in TB patients, wherein 88.2% and 81.1% sensitivities were obtained in pulmonary TB and extrapulmonary TB respectively, with 96-96.4% specificity. This assay may translate into a diagnostic kit.

Diagnosis of peritoneal tuberculosis by real-time immuno-PCR assay based on detection of a cocktail of Mycobacterium tuberculosis CFP-10 and HspX proteins.

BACKGROUND: Diagnosis of peritoneal TB is difficult owing to unusual clinical manifestations and low sensitivities obtained with most of the available diagnostic modalities. Hence, there is an urgent need to design a reliable diagnostic test so that an early therapy is initiated. RESEARCH DESIGN AND METHODS: We designed a quantitative real-time immuno-PCR (RT-I-PCR) assay to detect a cocktail of Mycobacterium tuberculosis CFP-10 (Rv3874) and HspX (Rv2031c) proteins in clinical samples (ascitic fluids and peritoneal biopsies) of peritoneal TB patients, and results were compared with I-PCR/ELISA. RESULTS: A wide range of CFP-10+ HspX (0.6 pg/mL to 9.9 ng/mL) was detected in clinical samples of peritoneal TB patients by RT-I-PCR, whereas ELISA exhibited a narrow range (3 ng/mL to 11.5 ng/mL). Sensitivities of 81.5% and 65.7% and specificities of 92.5% and 90% were obtained in a total of 78 cases (comprising 38 peritoneal TB and 40 non-TB controls) by RT-I-PCR and I-PCR, respectively. Markedly, sensitivity obtained by RT-I-PCR was significantly higher than I-PCR (p = 0.0143) and ELISA (p = 0.0005). CONCLUSIONS: Our RT-I-PCR revealed good accuracy for the rapid diagnosis of peritoneal TB cases. After further improving the specificity and reducing the cost, this assay may develop into a diagnostic kit.

Genome-wide analysis of PTR transporters in Candida species and their functional characterization in Candida auris.

The peptide transport (PTR) or proton-dependent oligopeptide transporter (POT) family exploits the inwardly directed proton motive force to facilitate the cellular uptake of di/tripeptides. Interestingly, some representatives are also shown to import peptide-based antifungals in certain Candida species. Thus, the identification and characterization of PTR transporters serve as an essential first step for their potential usage as antifungal peptide uptake systems. Herein, we present a genome-wide inventory of the PTR transporters in five prominent Candida species. Our study identifies 2 PTR transporters each in C. albicans and C. dubliniensis, 1 in C. glabrata, 4 in C. parapsilosis, and 3 in C. auris. Notably, despite all representatives retaining the conserved features seen in the PTR family, there exist two distinct classes of PTR transporters that differ in terms of their sequence identities and lengths of certain extracellular and intracellular segments. Further, we also evaluated the contribution of each PTR protein of the newly emerged multi-drug-resistant C. auris in di/tripeptide uptake. Notably, deletion of two PTR genes BNJ08_003830 and BNJ08_005124 led to a marked reduction in the transport capabilities of several tested di/tripeptides. However, all three genes could complement the role of native PTR2 gene of Saccharomyces cerevisiae, albeit to varied levels. Besides, BNJ08_005124 deletion also resulted in increased resistance toward the peptide-nucleoside drug Nikkomycin Z as well as the glucosamine-6-phosphate synthase inhibitor, L-norvalyl-N3-(4-methoxyfumaroyl)-L-2,3-diaminopropionoic acid (Nva-FMDP), pointing toward its predominant role in their uptake mechanism. Altogether, the study provides an important template for future structure-function investigations of PTR transporters in Candida species. KEY POINTS: • Candida genome encodes for two distinct classes of PTR transporters. • Candida auris encodes for 3 PTR transporters with different specificities. • BNJ08_005124 in C. auris is involved in the uptake of Nikkomycin Z and Nva-FMDP.

Identification of mycobacterial MPT-64 and ESAT-6 proteins in urogenital tuberculosis patients by real-time immuno-PCR.

Aim: Diagnosis of urogenital tuberculosis (UGTB) is difficult and there is an immediate need to develop a reliable diagnostic test. Methods: A real-time immuno-PCR (RT-I-PCR) was developed to identify a cocktail of MPT-64 + ESAT-6 in both male/female UGTB patients comprising five confirmed cases, 40 clinically suspected cases and 37 non-TB controls, from whom mid-stream urine specimens were collected, while endometrial biopsies of female patients were obtained on day 1 of their menstrual cycle. Results obtained by RT-I-PCR were compared with I-PCR/ELISA and GeneXpert. Results: A wide range (500 fg/ml-10 ng/ml) of MPT-64 + ESAT-6 was detected in UGTB specimens by RT-I-PCR, although ELISA showed a narrow range (2.5-11 ng/ml). Sensitivities of 80% and 82.2% were obtained by RT-I-PCR in clinically suspected and total UGTB cases, respectively, whereas 94.6% specificity was obtained. Concurrently, RT-I-PCR revealed significantly higher (p < 0.05-0.001) sensitivity than I-PCR/ELISA and GeneXpert. Conclusion: After improving the specificity, the authors may develop RT-I-PCR into a diagnostic kit.

Urogenital tuberculosis (UGTB) involves infection of the urinary tract and genital organs of male/female patients by Mycobacterium tuberculosis bacteria. Delayed diagnosis and therapy of UGTB lead to infertility and kidney failure. The routine tests used to detect the bacteria are not very sensitive due to low levels of bacteria present in UGTB specimens. Moreover, most nucleic acid amplification tests, such as PCR tests, give false-positive and false-negative results. The authors designed a real-time immuno-PCR test for detecting a cocktail of M. tuberculosis proteins in UGTB patients that revealed quite promising results, which were superior to immuno-PCR/ELISA and GeneXpert tests. After further improvement in the specificity and reduction of the price, this real-time immuno-PCR test could be used in routine diagnosis.

Spontaneous Suppressors against Debilitating Transmembrane Mutants of CaMdr1 Disclose Novel Interdomain Communication via Signature Motifs of the Major Facilitator Superfamily.

The Major Facilitator Superfamily (MFS) drug:H+ antiporter CaMdr1, from Candida albicans, is responsible for the efflux of structurally diverse antifungals. MFS members share a common fold of 12−14 transmembrane helices (TMHs) forming two N- and C-domains. Each domain is arranged in a pseudo-symmetric fold of two tandems of 3-TMHs that alternatively expose the drug-binding site towards the inside or the outside of the yeast to promote drug binding and release. MFS proteins show great diversity in primary structure and few conserved signature motifs, each thought to have a common function in the superfamily, although not yet clearly established. Here, we provide new information on these motifs by having screened a library of 64 drug transport-deficient mutants and their corresponding suppressors spontaneously addressing the deficiency. We found that five strains recovered the drug-resistance capacity by expressing CaMdr1 with a secondary mutation. The pairs of debilitating/rescuing residues are distributed either in the same TMH (T127ATMH1- > G140DTMH1) or 3-TMHs repeat (F216ATMH4- > G260ATMH5), at the hinge of 3-TMHs repeats tandems (R184ATMH3- > D235HTMH4, L480ATMH10- > A435TTMH9), and finally between the N- and C-domains (G230ATMH4- > P528HTMH12). Remarkably, most of these mutants belong to the different signature motifs, highlighting a mechanistic role and interplay thought to be conserved among MFS proteins. Results also point to the specific role of TMH11 in the interplay between the N- and C-domains in the inward- to outward-open conformational transition.

Early Time-Restricted Feeding Amends Circadian Clock Function and Improves Metabolic Health in Male and Female Nile Grass Rats.

Lengthening the daily eating period contributes to the onset of obesity and metabolic syndrome. Dietary approaches, including energy restriction and time-restricted feeding, are promising methods to combat metabolic disorders. This study explored the effect of early and late time-restricted feeding (TRF) on weight and adiposity, food consumption, glycemic control, clock gene expression, and liver metabolite composition in diurnal Nile grass rats (NGRs). Adult male and female Nile grass rats were randomly assigned to one of three groups: (1) access to a 60% high-fat (HF) diet ad-libitum (HF-AD), (2) time-restricted access to the HF diet for the first 6 h of the 12 h light/active phase (HF-AM) or (3) the second 6 h of the 12 h light/active phase (HF-PM). Animals remained on their respective protocols for six weeks. TRF reduced total energy consumption and weight gain, and early TRF (HF-AM) reduced fasting blood glucose, restored Per1 expression, and reduced liver lipid levels. Although sex-dependent differences were observed for fat storage and lipid composition, TRF improved metabolic parameters in both male and female NGRs. In conclusion, this study demonstrated that early TRF protocol benefits weight management, improves lipid and glycemic control, and restores clock gene expression in NGRs.