Characterization and in-vitro plant-based control of hindgut bacteria isolated from Odontotermes obesus Rambur (Termitidae) and Heterotermes indicola Wasmann (Rhinotermitidae).

Termites cause a serious menace to wooden structures all over the world. They rely mostly on entozoic fauna residing in their hindgut for the digestion of cellulosic and hemicellulosic materials. One of the ways to control termites is through their gut symbionts. The present study was designed to characterize the hindgut bacteria isolated from Odontotermes obesus and Heterotermes indicola. Furthermore, the growth inhibitory effect of eight tropical plant extracts was investigated to find out potential control agents for these bacterial isolates. The characterization of bacteria was carried out based on their morphology, Gram staining, biochemical and amplification of 16SrRNA gene. Amplified products were sequenced to confirm their relationship with bacterial isolates from termites of other regions. The growth inhibitory effect of ethanolic leaf extracts of eight plants was evaluated in an invitro agar well diffusion method. Qualitative and quantitative phytochemical analysis of the most effective plant was carried out to learn about bioactive agents. The results confirmed the presence of five bacteria from each termite species. The Bacillus cereus, Escherichia coli, and Lysinibacillus fusiformis were common to both termites whereas Lysinibacillus xylanilyticus and Lysinibacillus macrolides were found in O. obesus only and H. indicola harbor Bacillus subtilis and Shigella sonnei in addition to common three ones. Among the plant extracts of Carica papaya, Eucalyptus camaldulensis, Osmium basilicum, Grevillea robusta, Eucalyptus globulus, Pongamia pinnata, Mentha longifolia, and Melia azedarach, the G. robusta > E. camaldulensis > O. basilicum were found to have growth inhibitory effects with increasing concentrations from 100 to 2000 µg/mL. The biodiversity of the bacterial fauna is important for the biological control of termites. Leaf extracts of these medicinal plants can be used to control termite infestation in an environment-friendly manner to save huge economic loss.

The deubiquitinase USP40 preserves endothelial integrity by targeting the heat shock protein HSP90ß.

Endothelial cell (EC) barrier disruption and inflammation are the pathological hallmarks of vascular disorders and acute infectious diseases and related conditions, including the coronavirus disease 2019 (COVID-19) and sepsis. Ubiquitination plays a critical role in regulating the stability, intracellular trafficking, and enzymatic activity of proteins and is reversed by deubiquitinating enzymes (DUBs). The role of DUBs in endothelial biology is largely unknown. In this study, we report that USP40, a poorly characterized DUB, prevents EC barrier disruption through reductions in the activation of RhoA and phosphorylation of myosin light chain (MLC) and cofilin. Furthermore, USP40 reduces EC inflammation through the attenuation of NF-ĸB activation, ICAM1 expression, and leukocyte-EC adhesion. We further show that USP40 activity and expression are reduced in response to endotoxin challenge. Global depletion of USP40 and EC-targeted USP40 depletion in mice exacerbated experimental lung injury, whereas lentiviral gene transfer of USP40 protected against endotoxin-induced lung injury. Using an unbiased approach, we discovered that the protective effect of USP40 occurs through the targeting of heat shock protein 90ß (HSP90ß) for its deubiquitination and inactivation. Together, these data reveal a critical protective role of USP40 in vascular injury, identifying a unique mechanistic pathway that profoundly impacts endothelial function via DUBs.

Non-Lethal Doses of RSL3 Impair Microvascular Endothelial Barrier through Degradation of Sphingosie-1-Phosphate Receptor 1 and Cytoskeletal Arrangement in A Ferroptosis-Independent Manner.

The excess microvascular endothelial permeability is a hallmark of acute inflammatory diseases. Maintenance of microvascular integrity is critical to preventing leakage of vascular components into the surrounding tissues. Sphingosine-1-phosphate (S1P) is an active lysophospholipid that enhances the endothelial cell (EC) barrier via activation of its receptor S1PR1. Here, we delineate the effect of non-lethal doses of RSL3, an inhibitor of glutathione peroxidase 4 (GPX4), on EC barrier function. Low doses of RSL3 (50-100 nM) attenuated S1P-induced human lung microvascular barrier enhancement and the phosphorylation of AKT. To investigate the molecular mechanisms by which RSL3 attenuates S1P's effect, we examined the S1PR1 levels. RSL3 treatment reduced S1PR1 levels in 1 h, whereas the effect was attenuated by the proteasome and lysosome inhibitors as well as a lipid raft inhibitor. Immunofluorescence staining showed that RSL3 induced S1PR1 internalization from the plasma membrane into the cytoplasm. Furthermore, we found that RSL3 (100 and 200 nM) increased EC barrier permeability and cytoskeletal rearrangement without altering cell viability. Taken together, our data delineates that non-lethal doses of RSL3 impair EC barrier function via two mechanisms. RSL3 attenuates S1P1-induced EC barrier enhancement and disrupts EC barrier integrity through the generation of 4-hydroxynonena (4HNE). All these effects are independent of ferroptosis.

ISGylation of NF-κBp65 by SCFFBXL19 E3 Ligase Diminishes Endothelial Inflammation.

BACKGROUND: NF-κB (nuclear factor kappa B) plays a pivotal role in endothelial cell (EC) inflammation. Protein ISGylation is regulated by E3 ISG15 (interferon-stimulated gene 15) ligases; however, ISGylation of NF-κBp65 and its role in EC functions have not been investigated. Here, we investigate whether p65 is ISGylated and the role of its ISGylation in endothelial functions. METHODS: In vitro ISGylation assay and EC inflammation were performed. EC-specific transgenic mice were utilized in a murine model of acute lung injury. RESULTS: We find that NF-κBp65 is ISGylated in resting ECs and that the posttranslational modification is reversible. TNFα (tumor necrosis factor alpha) and endotoxin stimulation of EC reduce p65 ISGylation, promoting its serine phosphorylation through reducing its association with a phosphatase WIP1 (wild-type p53-induced phosphatase 1). Mechanistically, an SCF (Skp1-Cul1-F-box) protein E3 ligase SCFFBXL19 is identified as a new ISG15 E3 ligase that targets and catalyzes ISGylation of p65. Depletion of FBXL19 (F-box and leucine-rich repeat protein 19) increases p65 phosphorylation and EC inflammation, suggesting a negative correlation between p65 ISGylation and phosphorylation. Moreover, EC-specific FBXL19 overexpressing humanized transgenic mice exhibit reduced lung inflammation and severity of experimental acute lung injury. CONCLUSIONS: Together, our data reveal a new posttranslational modification of p65 catalyzed by a previously unrecognized role of SCFFBXL19 as an ISG15 E3 ligase that modulates EC inflammation.

Biocidal action, characterization, and molecular docking of Mentha piperita (Lamiaceae) leaves extract against Culex quinquefasciatus (Diptera: Culicidae) larvae.

Mosquitoes are found in tropical and subtropical areas and are the carriers of a variety of diseases that are harmful to people's health. e.g., malaria, filariasis, chikungunya, dengue fever, etc. Although several insecticides are available, however, due to insect resistance and environmental hazards, more eco-friendly chemicals are needed for insect control. So, the current research was planned to explore the prospective of Mentha piperita to be used for the formulation of larvicides against mosquito Culex quinquefasciatus. The ethanolic and water extracts of M. piperita leaves were prepared using the soxhlet apparatus. The extracts were dried and subjected to prepare five concentrations multiple of 80 ppm. Each concentration was applied for its larvicidal efficacy setting an experiment (in triplicate) in plastic containers of 1000 ml with extracts, 30 larvae of all four instars separately, and fed with dog biscuits along with controls. Observations were taken after each 12 hrs. till 72 hrs. The antioxidant perspective of M. piperita was determined by DPPH radical scavenging, total antioxidant capacity, and ferric reducing power assays. Using brine shrimp lethality bioactivity, the cytotoxic study was perceived. Standard techniques were used to classify the M. piperita extract using preliminary qualitative and quantitative phytochemicals, UV-Vis spectroscopy, FT-IR, and GC-MS analysis. M. piperita ethanolic leaves extract after 24 hrs. of exposure in 400 ppm showed 93% (LC50 = 208.976 ppm) mortality in ethanolic extract and 80% (LC90 = 246.900 ppm) in the water extract. In treated larvae, biochemical examination revealed a substantial (P<0.05) decrease in proteins, carbohydrates, and fat contents. The ethanol extract of M. piperita was the most efficient, killing brine shrimp nauplii in 50% to 90% of cases. TAC (125.4 3.5gAAE/mg DW) and FRP (378.1 1.0gAAE/mg DW) were highest in the ethanolic extract of M. piperita. The presence of medicinally active components such as alkaloids, carbohydrates, flavonoids, and others in M. piperita leaves extract in ethanol was discovered. The UV-Vis spectrum showed two peaks at 209.509 and 282.814 nm with the absorption of 2.338 and 0.796 respectively. The FT-IR consequences exhibited the occurrence of alcohols, alkanes, aldehyde, aromatic rings, ether linkage, ester, and halo- compounds. The GC-MS analysis according to peak (%) area and retention time showed ten phytochemicals consisting of six major and four minor compounds. Among all the compounds, 1, 2-benzene dicarboxylic acid, and 3-ethyl-5, 5-dimethyl -6-phenyl bound well to the NS3 protease domain with PDB ID: 2FOM. Hence, for the prevention of health hazards and mosquito control, M. Piperita is a potential source of chemicals for insecticide formulation.

Improving osteoporosis treatment rates in inpatients admitted with hip fracture: A healthcare improvement initiative in a tertiary referral hospital.

Objective: This healthcare improvement initiative was designed to increase inpatient osteoporosis treatment after hip fracture. Methods: A new protocol was developed by Geriatric Medicine and Endocrinology departments at a tertiary care hospital in Sydney. Its aim was to standardize assessment and treatment of osteoporosis in patients admitted with hip fracture. Eligible inpatients would receive intravenous zoledronic acid during their admission. A 6-month sample of hip fracture patients admitted after the protocol's implementation was compared to a group admitted before. Data collected included demographics, biochemistry, treatment rates, adverse effects, and admission survival. Results: There was a considerable increase in osteoporosis treatment after introducing the protocol. Before the protocol's introduction, none of 36 eligible patients received treatment. After the intervention 79% (23 out of 29) of eligible patients were treated.All treated patients had renal function and serum calcium levels checked post-infusion with no adverse outcomes. Eight patients developed flu-like symptoms within 24 h of the infusion. There were no instances of arrhythmias, ocular inflammation, or death. The cost per patient treated was AUD $87. Conclusion: Adopting a standardized protocol for osteoporosis treatment in patients admitted for hip fracture was effective in improving treatment rates whilst being relatively safe and inexpensive.

Molecular characterization and genetic diversity of cutaneous leishmaniasis from North Eastern Pakistan.

The genetic diversity of Leishmania spp. in North Eastern Pakistan remains undetermined despite increased cases of cutaneous leishmaniasis (CL). This study was designed to decipher the molecular characterization and genetic diversity of Leishmania spp. in North Eastern Pakistan. Out of 13761 CL suspected cases, 567 cases were microscopically positive and confirmed as Leishmania spp. by internal transcribed spacer (ITS) gene amplification through the PCR- RFLP technique. Further, isolates were directly sequenced to conduct phylogenetic analysis for genetic diversity. Among suspected CL cases, Mirpur showed the highest proportion of CL infection with 4.85% (118/2431) of the cases, while the Neelum district showed the lowest percentage at 3.29% (9/273). The slide positivity rate, annual blood examination rate, and annual parasite incidence rate were 3.84, 0.27, and 0.01% respectively, and the incidence of CL in the age group 1-20 years old was higher in males (50.92%) than females (25.75%). The RFLP analysis and sequencing confirmed the occurrence of Leishmania tropica, Leishmania major, and Leishmania infantum. Leishmania tropica (p = 0.02) confirmed significantly higher nucleotides variation than L. major (p = 0.05). Current findings confirmed the prior assumption that anthroponotic CL is the primary CL form present in North Eastern Pakistan. Moreover, this is the first report based on molecular identification of L. major, and L. infantum from North Eastern Pakistan. This remarkable heterogeneity in the Leishmania spp. is the leading cause of treatment failure and emergence of new haplotypes. Therefore more extensive investigations are recommended from all geographical regions of North Eastern Pakistan, especially those using a large sample size.

Spatiotemporal patterns of cutaneous leishmaniasis in the district upper and lower Dir, Khyber Pakhtunkhwa, Pakistan: A GIS-based spatial approaches.

While Cutaneous leishmaniasis (CL) is not a life-threatening disease, it leads to devastating effects on local community. CL is widely scattered manifesting a noticeable epidemiological pattern around the globe. The present study was planned to address the role of Geographic Information System (GIS) using CL clinico-epidemiological data to determine the high-risk areas of CL. Recorded data (2014-2018) of 3630 positive individuals was collected from Basic Health Units of the Upper and Lower Dir Districts, Khyber Pakhtunkhwa, Pakistan. Descriptive and statistical analysis was used for clinico-epidemiological characterization. For spatial analysis, ArcGIS V.10.3 was used and the CL average incidence was tagged on the proportional, choropleth, and digital elevation model maps. For focal transmission and high-risk zones, Inverse Density Weight (IDW) spatial interpolation, focal statistics, hot spot, cluster and outlier, and Bayesian geostatistical analysis were used. The trend of CL cases was elevated from 2014 to 2016 except for 2017 and 2018. Individuals of both genders younger than 20 years old were highly susceptible. Single lesions were more prominent (56%) and frequently affected facial parts (51%). The size and pretreatment duration of the CL lesion was significantly associated. Spatially, a choropleth map displayed the maximum CL incidences in Tehsil Balambat, Khal, and Termergara (31%-13%) located within a range of 948-1947m elevation in the central regions with proximal CL transmissions. Hot spot and cluster and outlier analysis affirmed that Tehsil Khal was the high-risk CL foci. The Bayesian geostatistical analysis revealed high temperature, less altitude, and annual precipitation as important risk factors. An increasing trend in CL transmission has become evident, affecting both genders and <20 years old children. GIS resolute the concealed CL hubs in the least elevated central regions which warrant the control strategies to restrict future epidemics.

Exploring the ability of dihydropyrimidine-5-carboxamide and 5-benzyl-2,4-diaminopyrimidine-based analogues for the selective inhibition of L. major dihydrofolate reductase.

To tackle leishmaniasis, search for efficient therapeutic drug targets should be pursued. Dihydrofolate reductase (DHFR) is considered as a key target for the treatment of leishmaniasis. In current study, we are interested in the design and synthesis of selective antifolates targeting DHFR from L. major. We focused on the development of new antifolates based on 3,4-dihydropyrimidine-2-one and 5-(3,5-dimethoxybenzyl)pyrimidine-2,4-diamine motif. Structure activity relationship (SAR) studies were performed on 4-phenyl ring of dihydropyrimidine (26-30) template. While for 5-(3,5-dimethoxybenzyl)pyrimidine-2,4-diamine, the impact of different amino acids (valine, tryptophan, phenylalanine, and glutamic acid) and two carbon linkers were explored (52-59). The synthesized compounds were assayed against LmDHFR. Compound 59 with the IC50 value of 0.10 µM appeared as potent inhibitors of L. major. Selectivity for parasite DHFR over human DHFR was also determined. Derivatives 55-59 demonstrated excellent selectivity for LmDHFR. Highest selectivity for LmDHFR was shown by compounds 56 (SI = 84.5) and 58 (SI = 87.5). Compounds Antileishmanial activity against L. major and L. donovani promastigotes was also performed. To explore the interaction pattern of the synthesized compounds with biological macromolecules, the docking studies were carried out against homology modelled LmDHFR and hDHFR targets.

Novel homozygous variant in the TPO gene associated with congenital hypothyroidism and mild-intellectual disability.

Congenital hypothyroidism (CH) is one of the most common hereditary disorders affecting neonates worldwide. CH is a multifactorial complex disorder and can be caused by either environmental factors or genetic factors. We studied one Pakistani family with segregating mutations in CH inherited in an autosomal recessive manner. Using whole-exome sequencing (WES), we found a novel homozygous missense variant (c.2315A>G; p.Tyr772Cys) in the thyroid peroxidase (TPO) gene. Different bioinformatics prediction tools and Sanger sequencing were performed to verify the identified variant. Our findings highlight the importance of this gene in causing CH and mild-intellectual disability (ID) in two affected brothers. WES is a convenient and useful tool for the clinical diagnosis of CH and other associated disorders.

In vitro anti-leishmanial activity of Prunus armeniaca fractions on Leishmania tropica and molecular docking studies.

Prunus armeniaca (L.) is a member of the Rosaceae, subfamily Prunoideae, shows anticancer, antitubercular, antimutagenic, antimicrobial, antioxidant, and cardioprotective activities. Here we fractionated the leaves extract of this highly medicinally important plant for antileishmanial activity. In the current study, the leaves extract was fractionated and characterized using column and thin layer chromatography by n-hexane, ethyl acetate, and methanol solvents. Twelve fractions were isolated and subjected for evaluation of their cytotoxicity and in vitro antileishmanial activity against promastigotes and amastigotes of Leishmania tropica. Among all fractions used, the fraction (F7) exhibited the strongest antileishmanial activity. The bioactive fraction was further characterized by spectroscopy (FTIR, UV-Vis), and GC-MS analysis. The in silico docking was carried out to find the active site of PTR1. All derived fractions exhibited toxicity in the safety range IC50 > 100 µg/ml. The fraction (F7) showed significantly the highest antipromastigotes activity with IC5011.48 ± 0.82 µg/ml and antiamastigotes activity with IC50 21.03 ± 0.98 µg/ml compared with control i.e. 11.60 ± 0.70 and 22.03 ± 1.02 µg/ml respectively. The UV-Vis spectroscopic analysis revealed the presence of six absorption peaks and the FTIR spectrum revealed the presence of alkane, aldehyde, carboxylic acid, thiols, alkynes, and carbonyls compounds The GC-MS chromatogram exhibited the presence of nine compounds: (a) benzeneethanol, alpha, beta dimethyl, (b)carbazic acid, 3-(1 propylbutylidene)-, ethyl ester, (c)1, 2-benzenedicarboxylic acid, diisooctyl ester, (d)benzeneethanamine a-methyl, (e)2aminononadecane, (f)2-heptanamine-5-methyl, (g)cyclobutanol, (h)cyclopropyl carbine, and (i)nitric acid, nonyl ester. Among all compounds, the 1, 2-benzenedicarboxylic acid, diisooctyl ester bound well to the PTR1 receptor. Fraction (F7) showed acceptable results with no cytotoxicity. However, in vivo studies are required in the future.

Molecular epidemiological survey of cutaneous leishmaniasis from Azad Jammu and Kashmir, Pakistan.

Cutaneous leishmaniasis (CL) is an emerging neglected tropical disease in Azad Jammu and Kashmir which is an underdeveloped area. Prevalence and parasite species identification are the key factors to control disease in a particular population, which were the objectives of the present study. Due to a lack of previous data, we performed a district-based active CL surveillance in 2018. The data of CL, suspected (n = 20,000) cases were analyzed statistically and identified the parasite species in microscopic positive cases by ITS1-PCR RFLP and also obtained accession numbers MN891719-28 from gene Bank. The phylogenetic tree was constructed using MEGA6 software. Out of 20,000 CL, suspected cases the highest rate of 4.02% (135/3360) of CL in Mirpur and the lowest 1.58% (8/505) in Neelum was reported. The slide positivity rate, annual parasite incidence rate and annual blood examination rate were 2.27 per 1000 population, 0.08 and 0.34%. The males were more infected 58.12% (297/511) than females 41.88% (214/511) and the age group of 1-20 years were found highly infected 82.78% (423/511) than 21-40 years 13.89% (71/511) and 41-60 years 3.33% (17/511) in the studied population. The patients 56.36% (288/511) had a single lesion whereas 29.35% (150/511) had two, only 10.76% (31/288) and 8% (12/150) were using bed nets. The patients 14.29% (73/511) had three or more lesions were not using bed nets. Only 27.98% (143/511) patients had received treatment, while 72.02% (368/511) didn't. Microscopically positive cases were found to be 2.56% (511/20,000) and ITS1-PCR positive cases were found to be 91.39% (467/511). The RFLP assay confirmed the presence of Leishmania tropica in 467 samples.

Prevalence and distribution of human Plasmodium infection in Federally Administrative Tribal Areas of Pakistan.

About 3.6 million Pashtun and over 1.5 million immigrants from Afghanistan live in the federally administered tribal areas (FATA) on the border between Pakistan's Khyber Pakhtunkhwa Province and southern Afghanistan. Although malaria cases are common in FATA, no detailed studies have yet been performed to reveal the actual status of malaria in the local population and epidemiological data are insufficient to elucidate the actual incidence. A malariometric survey of 691 patients of all ages and genders in seven agencies (districts) in FATA was carried out in 2013 using whole blood samples. Microscopically confirmed positive species were subjected to nested-PCR for the reconfirmation and detection of four species of Plasmodium causing human malaria. Of the 626 PCR positive cases, 81.1% were P. vivax, 13.8% P. falciparum and 4.9% mixed species containing both P. vivax and P. falciparum. P. malariae and P. ovale and were not found in any analysis. Sixty-five microscopic positive samples were identified as negative by PCR. The incidence of P. vivax ranged from 10.4% in Orakzai Agency to 22.8% in North Waziristan Agency. The prevalence of P. falciparum ranged from 1.3% in Orakzai Agency to 4.7% in North Waziristan, and Khyber Agency had the highest prevalence of 1.7% of mixed species. In FATA, P. vivax and P. falciparum are the main causative agents of malaria, while mixed species infections are also prevalent with varying transmission intensities. In addition, Estimates of malaria incidence shows that variation in the incidence, frequency and species composition of malarial parasites is high.

Structure based medicinal chemistry-driven strategy to design substituted dihydropyrimidines as potential antileishmanial agents.

In an attempt to explore novel and more potent antileishmanial compounds to diversify the current inhibitors, we pursued a medicinal chemistry-driven strategy to synthesize novel scaffolds with common pharmacophoric features of dihydropyrimidine and chalcone as current investigational antileishmanial compounds. Based on the reported X-ray structure of Pteridine reductase 1 (PTR1) from Leishmania major, we have designed a number of dihydropyrimidine-based derivatives to make specific interactions in PTR1 active site. Our lead compound 8i has shown potent in vitro antileishmanial activity against promastigotes of L. Major and Leishmania donovani with IC50 value of 0.47 µg/ml and 1.5 µg/ml respectively. The excellent in vitro activity conclusively revealed that our lead compound is efficient enough to eradicate both visceral and topical leishmaniasis. In addition, docking analysis and in silico ADMET predictions were also carried out. Predicted molecular properties supported our experimental analysis that these compounds have potential to eradicate both visceral and topical leishmaniasis.