Green synthesis of pectin-functionalized silver nanocomposites using Carpesium nepalense and evaluation its bactericidal kinetics and hepatoprotective mechanisms.

The present study reports the green synthesis of pectin-fabricated silver nanocomposites (Pectin-AgNPs) using Carpesium nepalense leaves extract, evaluating their bactericidal kinetics, in vivo hepatoprotective, and cytotoxic potentials along with possible mechanisms. GC/MS and LC/MS analyses revealed novel phytochemicals in the plant extract. The Pectin-AgNPs were characterized using UV/Vis, AFM, SEM, TEM, DLS, FTIR, and EDX techniques, showing a spherical morphology with a uniform size range of 50-110 nm. Significant antibacterial activity (P < 0.005) was found against four bacterial strains with ZIs of 4.1 ± 0.15 to 27.2 ± 3.84 mm. AFM studies revealed significant bacterial cell membrane damage post-treatment. At 0.05 mg/kg, the nanocomposites showed significant (P < 0.005) hepatoprotective activity in biochemical and histopathology analyses compared to the CCl4 control group. Pectin-AgNPs significantly reduced (P < 0.005) LDH, AST, ALT, ALP, and DB levels. qPCR analysis showed ameliorative effects on PPARs and Nrf2 gene expression, restoring gene alterations caused by CCl4 intoxication. In vivo acute toxicity studies confirmed low toxicity of Pectin-AgNPs in major organs. Pectin-AgNPs exhibited cytotoxic activity against HeLa cell lines at higher doses with an LC50 of 223.7 µg/mL. These findings demonstrate the potential of Pectin-AgNPs as promising antibacterial, hepatoprotective, and cytotoxic agents.

Thermoresponsive lipids engineered magnetic nanoparticles for spatiotemporal delivery of hesperidin to inflammatory sites in animal model.

Thermoresponsive nanoparticles are exploited as drug-delivery vehicles that release their payload upon increment in temperature. We prepared and characterized thermoresponsive lipid-anchored folic acid engineered magnetic nanoparticles (LP-HP-FANPs) that combine receptor-based targeting and thermoresponsive sustained release of hesperidin (HP) in response to endogenous inflammation site temperature. The progressive surface engineering of NPs was validated by FTIR analysis. Our LP-HP-FANPs had a particle size of 100.5 ± 1.76 nm and a zeta potential of 14.6 ± 2.65 mV. The HP encapsulation effectiveness of LP-HP-FANPs is around 91 ± 0.78%. AFM scans indicated that our modified nanoparticles were spherical. LP-HP-FANPs exhibit increased drug release (85.8% at pH 4.0, 50.9% at pH 7.0) at 40 °C. Animal studies showed no toxicity from nanoparticles. Compared to conventional drugs and HP, LP-HP-FANPs effectively decreased paw edema, cytokine levels, and total cell recruitment in thioglycollate-induced peritonitis (p < 0.05). LP-HP-FANPs substantially decreased cytokines compared to HP, HP-FA-NPs, and the standard medication (p < 0.05, p < 0.01, and p < 0.001). These findings imply that the synthesized HP-loaded formulation (LP-HP-FANPs) may be a potential anti-inflammatory formulation for clinical development.

Glycolipids biosurfactants production using low-cost substrates for environmental remediation: progress, challenges, and future prospects.

The production of renewable materials from alternative sources is becoming increasingly important to reduce the detrimental environmental effects of their non-renewable counterparts and natural resources, while making them more economical and sustainable. Chemical surfactants, which are highly toxic and non-biodegradable, are used in a wide range of industrial and environmental applications harming humans, animals, plants, and other entities. Chemical surfactants can be substituted with biosurfactants (BS), which are produced by microorganisms like bacteria, fungi, and yeast. They have excellent emulsifying, foaming, and dispersing properties, as well as excellent biodegradability, lower toxicity, and the ability to remain stable under severe conditions, making them useful for a variety of industrial and environmental applications. Despite these advantages, BS derived from conventional resources and precursors (such as edible oils and carbohydrates) are expensive, limiting large-scale production of BS. In addition, the use of unconventional substrates such as agro-industrial wastes lowers the BS productivity and drives up production costs. However, overcoming the barriers to commercial-scale production is critical to the widespread adoption of these products. Overcoming these challenges would not only promote the use of environmentally friendly surfactants but also contribute to sustainable waste management and reduce dependence on non-renewable resources. This study explores the efficient use of wastes and other low-cost substrates to produce glycolipids BS, identifies efficient substrates for commercial production, and recommends strategies to improve productivity and use BS in environmental remediation.

Design and Development of a Self-nanoemulsifying Drug Delivery System for Co-delivery of Curcumin and Naringin for Improved Wound Healing Activity in an Animal Model.

The present study endeavored to design and develop a self-nanoemulsifying drug delivery system to improve the solubility and dermatological absorption of curcumin and naringin. Curcumin and naringin-loaded self-nanoemulsifying drug delivery system formulations were developed using aqueous phase titration. Phase diagrams were used to pinpoint the self-nanoemulsifying drug delivery system zones. Tween 80 and Labrasol (surfactants), Transcutol (cosurfactant), and cinnamon oil were chosen from a large pool of surfactants, cosurfactants, and oils based on their solubility and greatest nano-emulsion region. Fourier transform infrared spectroscopy, zeta sizer, and atomic force microscopy were used to characterize the optimized formulations and test for dilution and thermodynamic stability. The optimized curcumin-naringin-self-nanoemulsifying drug delivery system demonstrated the following characteristics: polydispersity index (0.412 ± 0.03), % transmittance (97%), particle size (212.5 ± 05 nm), zeta potential (- 25.7 ± 1.80 mV) and having a smooth and spherical droplet shape, as shown by atomic force microscopy. The ability of their combined formulation to cure wounds was tested in comparison to pure curcumin suspension, empty self-nanoemulsifying drug delivery system, and standard fusidic acid. Upon topical administration, the optimized curcumin-naringin-self-nanoemulsifying drug delivery system demonstrated significant wound healing activity in comparison with a pure curcumin suspension, empty self-nanoemulsifying drug delivery system, and standard fusidic acid. Based upon this result, we assume that skin penetration was increased by using the optimized curcumin-naringin-self-nanoemulsifying drug delivery system with enhanced solubility.

Immunologic characterization of the Bm05br protein using the Rhipicephalus linnaei (Audouin, 1826) species as a tick model.

The identification and characterization of tick proteins allow us to discover new physiological targets useful for the development of tick control methods. Bm05br (Brazil Rhipicephalus microplus protein 05) is a protein with unknown function identified in the saliva of R. microplus. Rs05br (Brazil Rhipicephalus sanguineus protein 05), a protein with 99 % similarity to Bm05br, was identified in Rhipicephalus linnaei egg, larval, and nymphal stages, as well as in adult saliva. To improve the knowledge about both proteins, immunological characterization was performed, including antigenicity analysis, vaccination trials, and artificial feeding. The sequence and antigenicity analysis of Bm05br and Rs05br proteins showed that R. linnaei could serve as a tick model for cross-protection studies. The recombinant Bm05br protein was immunogenic. Anti-Bm05br antibodies recognized the homologous protein Rs05br in different stages, organs, and in the saliva of R. linnaei. Although rBm05br did not induce a protective response against infestation in R. linnaei in this study, further experiments could be developed taking into account new formulations and animal models for vaccination. These results also serve as a reference for future research on the function of these proteins in R. microplus and R. linnaei physiology, as well as other species of the genus Rhipicephalus.

Fraxinellone alleviates colitis-related intestinal fibrosis by blocking the circuit between PD-1+ Th17 cells and fibroblasts.

BACKGROUND: Excessive activation of colonic fibroblasts and differentiation of T helper 17 (Th17) cells are the key steps for intestinal fibrogenesis in the process of inflammatory bowel disease (IBD). Although both transforming growth factor-beta (TGF-ß)/Mothers Against Decapentaplegic Homolog (SMAD) 3-induced fibroblasts activation and interleukin (IL)-6/signal transducer and activator of transcription (STAT) 3-induced Th17 differentiation have been well studied, the crosstalk between fibroblasts and Th17 cells in the process of intestinal fibrogenesis needs to be unveiled. METHODS: In this study, the activation of colonic fibroblasts was induced with dextran sulfate sodium salt (DSS) and TGF-ß in vivo and in vitro respectively. P-SMAD3 and its downstream targets were quantified using RT-PCR, western blot and immunofluorescence. The differentiation of programmed death 1 (PD-1) + Th17 and activation of fibroblasts were quantified by FACS. PD-1+ Th17 cells and fibroblasts were co-cultured and cytokines in the supernatant were tested by ELISA. The anti-fibrosis effects of different chemical compounds were validated in vitro and further confirmed in vivo. RESULTS: The colonic fibroblasts were successfully activated by DSS and TGF-ß in vivo and in vitro respectively, as activation markers of fibroblasts (p-SMAD3 and its downstream targets such as Acta2, Col1a1 and Ctgf) were significantly increased. The activated fibroblasts produced more IL-6 compared with their inactivated counterparts in vivo and in vitro. The proinflammatory cytokine IL-6 induced PD-1+ Th17 differentiation and TGF-ß that in return promoted the activation of colonic fibroblasts. Fraxinellone inhibited TGF-ß+ PD-1+ Th17 cells via deactivating STAT3. CONCLUSIONS: The reciprocal stimulation constructed a circuit of PD-1+ Th17 cells and fibroblasts that accelerated the fibrosis process. Fraxinellone was selected as the potential inhibitor of the circuit of PD-1+ Th17 cells and fibroblasts in vivo and in vitro. Inhibiting the circuit of PD-1+ Th17 cells and fibroblasts could be a promising strategy to alleviate intestinal fibrosis.

A randomized, double-blind, positive-controlled, Phase-II clinical trial to evaluate efficacy and safety of Fuke Qianjin capsule in Pakistani patients with pelvic inflammatory disease.

Ethnopharmacological relevance: Pelvic inflammatory disease (PID) is a frequently occurring gynecological disorder mainly caused by the inflammation of a woman's upper genital tract. Generally, antibiotics are used for treating PID, but prolonged use poses potential risks of gut bacterial imbalance, bacterial resistance, super bacteria production, and associated adverse reactions. Traditional Chinese medicine (TCM) has shown unique advantages in various ailments and has received widespread clinical research attention. Fuke Qianjin (FUKE) capsule is an approved National Medical Products Administration (NMPA License No. Z20020024) Chinese herbal prescription that has been widely used individually or in combination with other Western medicines for the treatment of various gynecological inflammatory diseases, including chronic cervicitis, endometritis, and chronic PID. Aim: This clinical trial was designed to assess the safety and efficacy of FUKE capsule in mild-to-moderate symptomatic PID patients. Materials and methods: This phase 2, randomized, double-blind, positive controlled clinical trial was conducted in mild-to-moderate symptomatic PID patients at a single center in Pakistan from 21 September 2021 to 11 March 2022. Eligible female participants were randomly assigned to a test and a control group with a ratio of 1:1. The test group subjects received two metronidazole (METRO) tablets and one doxycycline hyclate (DOXY) simulant at a time, twice daily for 14 days, and two Fuke Qianjin (FUKE) capsules, three times a day after a meal for 28 days. Subjects in the control group received two METRO tablets and one DOXY tablet at a time, twice daily for 14 days, and two FUKE simulant capsules, three times a day after meal for 28 days. The primary efficacy outcome was an improvement in pelvic pain symptoms assessed through a visual analog scale (VAS). The secondary outcomes were the improvement in secondary efficacy symptoms like local physical signs, clinical assessment of leucorrhea and cervical secretions through laboratory examination, and improvement in the maximum area of pelvic effusion assessed through gynecological ultrasound after the treatment. The safety outcomes were assessed through vital signs, laboratory tests, electrocardiogram findings, and adverse events/serious adverse events. Results: A total of 198 subjects with active PID were randomly assigned to a test group (n = 99) and a control group (n = 99). The baseline characteristics of the subjects in the two groups were similar. In the intention-to-treat analysis, the primary efficacy was 84.9% for the test group and 71.6% for the control group, with a statistically significant difference (p = 0.0370; 95% CI -0.2568 to -0.0088). The secondary clinical efficacy was 88.4% for the test group and 82.7% for the control group, with no significant difference (p = 0.2977; 95% CI -0.1632 to 0.0501). The improvement in local physical signs was 95.8% for the test group and 76.9% for the control group, with no significant difference (p = 0.0542; 95% CI -0.3697 to -0.0085). The inter-group non-inferiority comparison showed that the upper limit of the 95% CI was less than 0.15 and thus met the non-inferiority requirements of the test group to the control group. The results of clinical signs of leucorrhea and cervical secretions showed that there was no difference in the rate of improvement between the test and control groups, indicating that FUKE was non-inferior to DOXY. A total of 14 adverse events in eight subjects were observed in the trial, with an incidence rate of 4.7%. Four subjects in each group experienced seven adverse events with 4.5% and 4.8% incidence rates of adverse reactions in the test and control groups, with no statistically significant differences (p = 0.2001). No serious adverse events occurred in the trial. Conclusion: The results of this trial indicate that the test drug (Fuke Qianjin capsule) is non-inferior to the control drug (doxycycline hyclate tablet) in treating mild-to-moderate PID patients with comparable efficacy, safety, and tolerability to the control drug. Clinical Trial Registration: www.clinicaltrials.gov, identifier NCT04723069.

MDSC-targeted liposomal all-trans retinoic acid suppresses mMdscs and improves immunotherapy in HBV infection.

BACKGROUND: Myeloid-derived suppressor cells (MDSCs) are evolving as a prominent determinant in cancer occurrence and development and are functionally found to suppress T cells in cancer. Not much research is done regarding its involvement in viral infections. This research was designed to investigate the role of MDSCs in hepatitis B virus (HBV) infection and how targeting these cells with our novel all-trans retinoic acid encapsulated liposomal formulation could improve immunotherapy in C57BL/6 mice. METHODS: Ten micrograms (10 µg) of plasmid adeno-associated virus (pAAV/HBV 1.2, genotype A) was injected hydrodynamically via the tail vein of C57BL/6 mice. An all-trans retinoic acid encapsulated liposomal formulation (L-ATRA) with sustained release properties was used in combination with tenofovir disoproxil fumarate (TDF), a nucleotide analog reverse transcriptase inhibitor (nRTI) to treat the HBV infection. The L-ATRA formulation was given at a dose of 5 mg/kg intravenously (IV) twice a week. The TDF was given orally at 30 mg/kg daily. RESULTS: Our results revealed that L-ATRA suppresses MDSCs in HBV infected mice and enhanced T-cell proliferation in vitro. In vivo studies showed higher and improved immunotherapeutic effect in mice that received L-ATRA and TDF concurrently in comparison with the groups that received monotherapy. Lower HBV DNA copies, lower concentrations of HBsAg and HBeAg, lower levels of ALT and AST and less liver damage were seen in the mice that received the combination therapy of L-ATRA + TDF. CONCLUSIONS: In effect, targeting MDSCs with the combination of L-ATRA and TDF effectively reduced mMDSC and improved immunotherapy in the HBV infected mice. Targeting MDSCs could provide a breakthrough in the fight against hepatitis B virus infection.

An intrinsic electrical conductivity study of perovskite powders MAPbX3 (X = I, Br, Cl) to investigate its effect on their photovoltaic performance.

An investigation into the intrinsic electrical conductivity of perovskite powders MAPbX3, where X represents iodine (I), bromine (Br), or chlorine (Cl), was conducted to explore its impact on their photovoltaic performance. Results revealed that MAPbCl3 demonstrated light absorption ability in the ultraviolet and visible regions, while MAPbBr3 showed capacity for light absorption at longer wavelengths in the visible spectrum. On the other hand, MAPbI3 exhibited good absorption at longer wavelengths, indicating its ability to absorb light in the near-infrared region. The optical bandgap of each perovskite was determined to be 2.90 eV for MAPbCl3, 2.20 eV for MAPbBr3, and 1.47 eV for MAPbI3. The electrical conductivities of these powders were measured in-plane using the four-probe method and through-plane by electrochemical impedance spectroscopy (EIS). Electrochemical impedance spectroscopy (EIS) studies revealed a significant change in the conductivity of the MAPbI3 perovskite at temperatures between 80 °C and 100 °C. This change could be attributed to structural modifications induced when the temperature exceeds these values. The through-plane conductivity changed from 3 × 10-8 S cm-1 at 60 °C to approximately 6 × 10-5 S cm-1 at 120 °C and around 2 × 10-3 S cm-1 at 200 °C. Meanwhile, the sheet conductivity (in-plane conductivity) measurements performed at ambient temperature reveal that sheet conductivities are 489 × 103 S m-1, 486 × 103 S m-1 and 510 × 103 S m-1 for MAPbBr3, MAPbCl3 and MAPbI3, respectively. This study provides valuable insights for optimizing the performance of perovskite solar cells. Understanding how dopants influence the electrical conductivity and photovoltaic properties of the perovskite material, this work will enable researchers to design and engineer more efficient and stable solar cell devices based on MAPbX3 perovskites.

Nanocarrier-based localized and effective treatment of renal disorders: currently employed targeting strategies.

Renal disorders pose a global health threat, with targeted drug-delivery systems emerging as a promising strategy to enhance therapy safety and efficacy. Recent efforts have harnessed targeted nanomaterials for kidney disease treatment. While some systems remain in the early stages, they show immense potential in delivering cargo to specific sites. Through animal model experimentations, it has been demonstrated to reduce systemic side effects and enhance treatment effectiveness. This review presents current strategies for kidney disorder treatment, emphasizing site-specific targeting critical to renal disease pathophysiology. Recent advancements in nano-drug delivery systems for kidney targeting are explored. Finally, toxicological aspects and prospects of the most promising kidney-targeting delivery systems are discussed in this review article.

First Molecular-Based Confirmation of Dermacentor marginatus and Associated Rickettsia raoultii and Anaplasma marginale in the Hindu Kush Mountain Range.

Ticks of the genus Dermacentor Koch, 1844 (Acari: Ixodidae) are poorly known systematically due to their habitation in harsh topographic environments and high mountains. Dermacentor ticks are diversely distributed in the Palearctic, Nearctic, and Oriental regions. There is no available information on the occurrence of Dermacentor marginatus in Pakistan; thus, the current investigation aimed the first morphological and molecular confirmation of this species and associated Anaplasma marginale and Rickettsia raoultii. Ticks were collected from goats (Capra hircus) and morphologically identified. Genomic DNA was extracted from 18/26 (69.23%) tick specimens, including 11 males and 7 females (1 unfed and 6 fed females). Extracted DNA was subjected to PCR for the amplification of genetic markers like 16S rDNA and cox1 for ticks, 16S rDNA for Anaplasma spp., and gltA and ompB for Rickettsia spp. A total of 26 D. marginatus ticks composed of 19 males (73.07%) and 7 females (26.9%) [1 (3.84%) unfed and 6 (23.07%) fed females] were collected from goats. According to amplicons via BLAST analysis, the 16S rDNA sequence showed 97.28-98.85% identity and the cox1 sequence showed 95.82-98.03% identity with D. marginatus. Additionally, the 16S rDNA sequence for Anaplasma sp. was detected in D. marginatus that showed 100% identity with Anaplasma marginale. Rickettsial gltA and ompB sequences for Rickettsia sp. showed 100% identity with Rickettsia raoultii. In phylogenetic analysis, ticks' 16S rDNA and cox1 sequences clustered with the same species. In phylogenetic analysis, A. marginale based on 16 rDNA clustered with A. marginale, while gltA and ompB sequences clustered with R. raoultii. This is the first study on the genetic characterization of D. marginatus and associated A. marginale and R. raoultii in Pakistan. The northern areas of Pakistan, which need to be explored in terms of ticks and associated pathogens due to their zoonotic threats, have been neglected due to the inaccessible climatic conditions.

Green Synthesis of ZnO and Black TiO2 Materials and Their Application in Photodegradation of Organic Pollutants.

ZnO and black TiO2 have been selected as the most efficient materials for organic pollution abatement due to their increased efficiency when compared to other materials. However, the concept of green chemistry makes it desirable to design green synthesis approaches for their production. In this study, black TiO2 was synthesized using an environmentally safe synthetic technique with glycerol as a reductant. ZnO was prepared by using ionic-liquid-based microwave-assisted extracts of Polygonum minus. To investigate the materials' potential to photodegrade organic pollutants, methylene blue (MB) and phenol were chosen as model organic pollutants. Both materials were found to exhibit spherical morphologies and a mesoporous structure and were efficient absorbers of visible light. ZnO exhibited electron-hole pair recombination lower than that of black TiO2. Black TiO2 was discovered to be an anatase phase, whereas ZnO was found to have a hexagonal wurtzite structure. In contrast to black TiO2, which had a surface area of 239.99 m2/g and a particle size of 28 nm, ZnO had a surface area of 353.11 m2/g and a particle size of 32 nm. With a degradation time of 60 min, ZnO was able to eliminate 97.50% of the 40 mg/L MB. Black TiO2, on the other hand, could reduce 90.0% of the same amount of MB in 60 min. When tested for phenol degradation, ZnO and black TiO2 activities were reduced by nearly 15 and 25%, respectively. A detailed examination of both ZnO and black TiO2 materials revealed that ZnO has more potential and versatility for the degradation of organic pollutants under visible light irradiation.

First report on tick-borne pathogens detected in ticks infesting stray dogs near butcher shops.

Public health is a major concern for several developing countries due to infectious agents transmitted by hematophagous arthropods such as ticks. Health risks due to infectious agents transmitted by ticks infesting butcher-associated stray dogs (BASDs) in urban and peri-urban regions have been neglected in several developing countries. To the best of the authors' knowledge, this is the first study assessing public health risks due to ticks infesting BASDs in Pakistan's urban and peri-urban areas. A total of 575 ticks (390 from symptomatic and 183 from asymptomatic BASDs) were collected from 117 BASDs (63 symptomatic and 54 asymptomatic); the ticks belonged to 4 hard tick species. A subset of each tick species' extracted DNA was subjected to polymerase chain reaction (PCR) to amplify the 16S rDNA and cox1 sequences of the reported tick species, as well as bacterial and protozoal agents. The ticks' 16S rDNA and cox1 sequences showed 99-100% identities, and they were clustered with the sequence of corresponding species from Pakistan and other countries in phylogenetic trees. Among the screened 271 ticks' DNA samples, Anaplasma spp. was detected in 54/271 (19.92%) samples, followed by Ehrlichia spp. (n = 40/271, 14.76%), Rickettsia spp. (n = 33/271, 12.17%), Coxiella spp. (n = 23/271, 4.48%), and Hepatozoon canis (n = 9/271, 3.32%). The obtained sequences and phylogenetic analyzes revealed that the pathogens detected in ticks were Ehrlichia minasensis, Ehrlichia sp., Hepatozoon canis, Coxiella burnetii, Coxiella sp., Anaplasma capra, Anaplasma platys, Anaplasma sp., Rickettsia massiliae, "Candidatus Rickettsia shennongii" and Rickettsia aeschlimannii. Tick-borne pathogens such as E. minasensis, H. canis, A. capra, A. platys, and R. aeschlimannii, were detected based on the DNA for the first time in Pakistan. This is the first report on public health risks due to ticks infesting BASDs. These results not only provided insights into the occurrence of novel tick-borne pathogens in the region but also revealed initial evidence of zoonotic threats to both public health and domestic life.

Rabbits as Animal Models for Anti-Tick Vaccine Development: A Global Scenario.

Studies evaluating candidate tick-derived proteins as anti-tick vaccines in natural hosts have been limited due to high costs. To overcome this problem, animal models are used in immunization tests. The aim of this article was to review the use of rabbits as an experimental model for the evaluation of tick-derived proteins as vaccines. A total of 57 tick proteins were tested for their immunogenic potential using rabbits as models for vaccination. The most commonly used rabbit breeds were New Zealand (73.8%), Japanese white (19%), Californians (4.8%) and Flemish lop-eared (2.4%) rabbits. Anti-tick vaccines efficacy resulted in up to 99.9%. Haemaphysalis longicornis (17.9%) and Ornithodoros moubata (12.8%) were the most common tick models in vaccination trials. Experiments with rabbits have revealed that some proteins (CoAQP, OeAQP, OeAQP1, Bm86, GST-Hl, 64TRP, serpins and voraxin) can induce immune responses against various tick species. In addition, in some cases it was possible to determine that the vaccine efficacy in rabbits was similar to that of experiments performed on natural hosts (e.g., Bm86, IrFER2, RmFER2, serpins and serine protease inhibitor). In conclusion, results showed that prior to performing anti-tick vaccination trials using natural hosts, rabbits can be used as suitable experimental models for these studies.

Molecular detection of Coxiella spp. in ticks (Ixodidae and Argasidae) infesting domestic and wild animals: with notes on the epidemiology of tick-borne Coxiella burnetii in Asia.

Tick-borne Coxiella spp. are emerging in novel regions infecting different hosts, but information regarding their occurrence is limited. The purpose of this study was the molecular screening of Coxiella spp. in various ticks infesting goats, sheep, camels, cattle, wild mice, and domestic fowls (Gallus gallus domesticus) in various districts of Khyber Pakhtunkhwa, Pakistan. Morphologically identified tick species were confirmed by obtaining their cox1 sequences and were molecularly screened for Coxiella spp. by sequencing GroEL fragments. Almost 345 out of 678 (50.9%) hosts were infested by nine tick species. Regarding the age groups, the hosts having an age >3 years were highly infested (192/345, 55.6%), while gender-wise infestation was higher in female hosts (237/345, 68.7%). In collected ticks, the nymphs were outnumbered (613/1,119, 54.8%), followed by adult females (293/1,119, 26.2%) and males (213/1,119, 19.7%). A total of 227 ticks were processed for molecular identification and detection of Coxiella spp. The obtained cox1 sequences of nine tick species such as Hyalomma dromedarii, Hyalomma anatolicum, Haemaphysalis cornupunctata, Haemaphysalis bispinosa, Haemaphysalis danieli, Haemaphysalis montgomeryi, Rhipicephalus haemaphysaloides, Rhipicephalus microplus, and Argas persicus showed maximum identities between 99.6% and 100% with the same species and in the phylogenetic tree, clustered to the corresponding species. All the tick species except Ha. danieli and R. microplus were found positive for Coxiella spp. (40/227, 17.6%), including Coxiella burnetii (15/40, 6.7%), Coxiella endosymbionts (14/40, 6.3%), and different Coxiella spp. (11/40, 4.9%). By the BLAST results, the GroEL fragments of Coxiella spp. showed maximum identity to C. burnetii, Coxiella endosymbionts, and Coxiella sp., and phylogenetically clustered to the corresponding species. This is the first comprehensive report regarding the genetic characterization of Coxiella spp. in Pakistan's ticks infesting domestic and wild hosts. Proper surveillance and management measures should be undertaken to avoid health risks.

Characteristics and potential clinical applications of the extracellular vesicles of human pathogenic Fungi.

Extracellular vesicles (EVs) are a heterogeneous group of lipid membrane-enclosed compartments that contain different biomolecules and are released by almost all living cells, including fungal genera. Fungal EVs contain multiple bioactive components that perform various biological functions, such as stimulation of the host immune system, transport of virulence factors, induction of biofilm formation, and mediation of host-pathogen interactions. In this review, we summarize the current knowledge on EVs of human pathogenic fungi, mainly focusing on their biogenesis, composition, and biological effects. We also discuss the potential markers and therapeutic applications of fungal EVs.

First Report of Rickettsia conorii in Hyalomma kumari Ticks.

As a vector of wide range of pathogenic agents, ticks pose health threats to wild and domestic animals, and humans. Information is unavailable about the prevalence and spatial survey of Hyalomma kumari ticks and associated Rickettsia spp. in Pakistan. Concerning this knowledge gap, the present study aimed to molecularly detect Rickettsia species associated with H. kumari infesting small ruminants in Khyber Pakhtunkhwa (KP), Pakistan. A total of 409 H. kumari ticks were collected from 163/295 infested hosts with an infestation rate of 55.25%. A total of 204 females, 158 males, and 47 nymphs were collected. Goats were heavily infested by 224 ticks having an infestation rate of 58.33% (98/168), whereas sheep were infested by 185 ticks having a lesser infestation rate of 51.18% (65/127). Genomic DNA extracted from ticks was used for the amplification of tick (cox I, 16S rRNA, ITS-2) species and Rickettsia (gltA, ompA, and ompB) partial genes. Eighty-three ticks were subjected to PCR, and 8/83 (9.6%) were found positive for rickettsial agents. The cox I and 16S rRNA sequences of H. kumari showed 98.90-99.74% identity with H. kumari sequences reported from Pakistan, and phylogenetically clustered to the corresponding species reported from Pakistan and India. The obtained rickettsial gltA, ompA, and ompB sequences showed 100% identity with Rickettsia sp. of the Rickettsia conorii reported from Pakistan. In the phylogenetic trees, rickettsial sequences clustered with uncharacterized Rickettsia sp. from Pakistan and R. conorii from Israel, Russia, South Africa, and India. The present molecular based detection of H. kumari-associated R. conorii will facilitate effective surveillance in the region.

Phylogenetic Position of Haemaphysalis kashmirensis and Haemaphysalis cornupunctata, with Notes on Rickettsia spp.

Despite high diversity in the Oriental region, ticks of the genus Haemaphysalis have been neglected regarding their genetic data and vector potential. This study aimed to genetically characterize three species of the genus Haemaphysalis: Haemaphysalis cornupunctata, Haemaphysalis kashmirensis, and Haemaphysalis montgomeryi infesting goats and sheep, and Rickettsia spp. associated with these tick species in the Hindu Kush Himalayan range of Pakistan. Altogether, 834 ticks were collected by examining 120 hosts including goats (64/120, 53.3%) and sheep (56/120, 46.6%), in which 86 (71.6%) hosts were found to be tick-infested. The morphologically identified ticks were subjected to DNA extraction and PCR for the amplification of partial 16S rDNA and cox fragments. Rickettsia spp. associated with the collected ticks were detected through the amplification of gltA, ompA and ompB partial fragments. The 16S rDNA of H. cornupunctata and H. montgomeryi showed a maximum identity of 100% with the sequences of the same species, whereas the 16S rDNA of H. kashmirensis showed the highest identity of 93-95% with Haemaphysalis sulcata. The cox sequence of H. montgomeryi displayed 100% identity with the same species. In comparison, the cox sequences of H. cornupunctata and H. kashmirensis showed maximum identities of 87.65-89.22% with Haemaphysalis punctata and 89.34% with H. sulcata, respectively. The gltA sequence of Rickettsia sp. from H. kashmirensis showed the highest identity of 97.89% with Rickettsia conorii subsp. raoultii, while the ompA and ompB fragments from the same DNA samples revealed 100% and 98.16% identity with Rickettsia sp. and "Candidatus Rickettsia longicornii", respectively. Another gltA sequence amplified from H. montgomeryi ticks showed 100% identity with Rickettsia hoogstraalii, while the attempts to amplify ompA and ompB for R. hoogstraalii were unsuccessful. In the phylogenetic tree, the 16S rDNA of H. cornupunctata clustered with the corresponding species while its cox clustered with H. punctata. Both 16S rDNA and cox sequences of H. kashmirensis clustered with H. sulcata. The gltA sequence of Rickettsia sp. was clustered individually in the spotted fever (SF) group of Rickettsia, while the gltA sequence of R. hoogstraalii was clustered with the same species in the transition group of Rickettsia. In the SF group, the rickettsial ompA and ompB sequence clustered with undetermined Rickettsia sp. and "Candidatus Rickettsia longicornii", respectively. This is the earliest study regarding the genetic characterization of H. kashmirensis. This study indicated that ticks belong to the genus Haemaphysalis have the potential of harboring and/or transmitting Rickettsia spp. in the region.

Validation of catalytic site residues of Ubiquitin Specific Protease 2 (USP2) by molecular dynamic simulation and novel kinetics assay for rational drug design.

Post-translational modifications of proteins such as protein ubiquitination are crucial for regulating conformation, stability and localization of the modified protein. Ubiquitin-specific protease 2 (USP2), a multifunctional cysteine protease is reported to be a key regulator of ubiquitylation events in numerous oncogenic proteins e.g., fatty acid synthetase, Mdm2, EGFR, cyclin A1, and cyclin-D1, etc. Thus targeting USP2 is a promising strategy for cancer therapy. USP2 is characterized by a catalytic triad comprising of cysteine, histidine and aspartic acid residues. Five residues including three from the catalytic triad and two from outside of the catalytic triad have been reported as a catalytic site of USP2 that catalyze hydrolysis and stabilizes the oxyanion formed in the intermediate step of catalysis. Here, we report two more novel residues (L269 and Y558) on USP2 involved in the catalysis of Ubiquitin using computational alanine scanning (CAS) followed by molecular dynamic simulation studies. The results obtained from CAS were further validated by a highly reliable, time- and cost-effective SDS-PAGE-based kinetics assay using UBA52 which is a natural substrate of USP2. Our results showed that mutating L269 and Y558 significantly compromised the catalytic efficiency of USP2 in hydrolyzing UBA52 which can further be extended to rational drug design of USP2 selective inhibitors and to explore the catalytic sites of other USPs. Two novel residues take part in catalytic activity of USP2 which were depicted by MD Simulations and were further validated by novel SDS-PAGE-based reliable time- and cost-effective kinetics assay.

Synthetic star shaped tetra-tailed biocompatible supramolecular amphiphile as an efficient nanocarrier for Amphotericin B.

Macrocycle-based amphiphiles are capable of self-assembling into multidimensional nano-architecture with defined dimensions for various applications. Herein we report the synthesis, physio-chemical characterizations and oral drug delivery profiling of resorcinarene-based amphiphilic supramolecular macrocycle. The macrocycle was synthesized in two-step reaction and characterized using 1H NMR, Mass spectrometry and IR spectroscopic techniques. The synthesized macrocycle was assessed for vesicles formation, checked for biocompatibility and then Amphotericin B (Amp-B) was entrapped in macrocycle-based vesicles. The drug loaded vesicles were characterized for shape, size, homogeneity, drug entrapment, surface charge, in-vitro release profile and stability. Amp-B loaded macrocycle based vesicles were examined in rabbits for in-vivo bioavailability and compared with plan drug suspension. The synthesized macrocycle was non-toxic in normal mouse fibroblast cells, compatible with blood and safe in mice. The drug loaded macrocycle based vesicles appeared spherical with 279.4 nm size and - 12.2 mV zeta potential loading 85.45 % drug. The drug loaded vesicles storage stability for 30 days and gastric fluid stability for 1 h were it retained nearly 90 % drug at 30th day and 83.79 % drug at 1 h in gastric fluid. Oral bioavailability of Amp-B in rabbits was markedly enhanced when delivered in synthesized macrocycle based vesicles in comparison with plan drug suspension. Results of this study indicate that the synthesized star shaped tetra-tailed supramolecular amphiphile could be used as an efficient nanocarrier for enhancing oral bioavailability of drugs with solubility and bioavailability issues like Amp-B.