Evaluation of HPV E6/E7 mRNA Detection in Clinically Suspected Cases of Cervical Cancer with Abnormal Cytology: Time to Upgrade the Screening Protocols.

Background Human papillomavirus (HPV) E6/E7 mRNA tests determine the oncogenic activity of the virus and represent a good clinical biomarker for predicting the risk of cervical cancer. So, the present study was conducted to know the role of HPV E6/E7 mRNA as a predictive biomarker for cervical carcinoma. Methodology The present study was conducted on 55 clinical samples of cervical scrapings and biopsy from the clinically suspected cases (based on signs and symptoms) of cervical cancer having abnormal PAP smear. The samples were processed in three steps-(1) HPV DNA detection, (2) HPV E6/E7 mRNA detection, and (3) histopathological analysis. Results Out of a total of 55 patients, 16 (29.09%) were positive for both HPV E6/E7 mRNA and HPV DNA and six were positive for only HPV DNA. So, a total of 22 (40%) patients were positive for HPV DNA. Out of these 22 samples, 10 (45.5%) were of HPV-16, six (27.3%) were of HPV-18, four (18.2%) were of HPV-31, and two (9.1%) were of HPV-45. Out of total 16 patients positive for HPV E6/E7 mRNA, 10 (62.5%) were of genotype 16 and six (37.5%) were of genotype 18. The patients who were found positive for HPV 31 and 45 genotypes did not have E6/E7 mRNA expression. On colposcopic-guided biopsy, among these 16 samples, eight (50%) were diagnosed with invasive squamous cell carcinoma, six (37.5%) with cervical intraepithelial neoplasia grade 3 (CIN3), and two (12.5%) with CIN2. Out of those six patients in whom only HPV DNA was positive, five had normal biopsy findings and one had CIN1. Conclusion The present study suggests that HPV E6/E7 mRNA detection could be more reliable than DNA testing for predicting the risk of progression of HPV-induced cervical lesions to cervical carcinoma and it can be used as a non-invasive tool for triage and patient follow-up.

Noisy Galvanic Vestibular Stimulation Combined With a Multisensory Balance Program in Older Adults With Moderate to High Fall Risk: Protocol for a Feasibility Study for a Randomized Controlled Trial.

BACKGROUND: Reduced mobility and falls are common among older adults. Balance retraining programs are effective in reducing falls and in improving balance and mobility. Noisy galvanic vestibular stimulation is a low-level electrical stimulation used to reduce the threshold for the firing of vestibular neurons via a mechanism of stochastic resonance. OBJECTIVE: This study aims to determine the feasibility of using noisy galvanic vestibular stimulation to augment a balance training program for older adults at risk of falls. We hypothesize that noisy galvanic vestibular stimulation will enhance the effects of balance retraining in older adults at risk of falls. METHODS: In this 3-armed randomized controlled trial, community dwelling older adults at risk of falling will be randomly assigned to a noisy galvanic vestibular stimulation plus balance program (noisy galvanic vestibular stimulation group), sham plus balance program (sham group), or a no treatment group (control). Participants will attend the exercise group twice a week for 8 weeks with assessment of balance and gait pretreatment, posttreatment, and at 3 months postintervention. Primary outcome measures include postural sway, measured by center of pressure velocity, area and root mean square, and gait parameters such as speed, step width, step variability, and double support time. Spatial memory will also be measured using the triangle completion task and the 4 Mountains Test. RESULTS: Recruitment began in November 2020. Data collection and analysis are expected to be completed by December 2022. CONCLUSIONS: This study will evaluate the feasibility of using noisy galvanic vestibular stimulation alongside balance retraining in older adults at risk of falls and will inform the design of a fully powered randomized controlled trial. TRIAL REGISTRATION: New Zealand Clinical Trials Registry (ACTRN12620001172998); https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=379944. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/32085.

Encapsulating Au-Fe3 O4 Nanodots into AIE-active Supramolecular Assemblies: Ambient Visible-light Harvesting "Dip-Strip" Photocatalyst for C-C/C-N Bond Formation Reactions.

The present study demonstrates the development of a supramolecular porous ensemble consisting of hetero-oligophenylene derivative 6 and Au-Fe3 O4 nanodots. Supramolecular assemblies of AIE-active hetero-oligophenylene derivative 6 served as reactors for the generation of Au-Fe3 O4 nanodots. The as prepared supramolecular ensemble functioned as an efficient recyclable photocatalytic system for C(sp2 )-H bond activation of anilines for the construction of quinoline carboxylates. Interestingly, the "dip catalyst" prepared by depositing PTh-co-PANI-6: Au-Fe3 O4 nanodots on a filter paper served as a recyclable strip (up to 10 cycles) for C-C/C-N bond formation reaction.

UV light promoted 'Metal'/'Additive'-free oxidation of alcohols: investigating the role of alcohols as electron donors.

UV light promoted selective oxidation of primary and secondary alcohols has been demonstrated under 'metal-free' and 'additive-free' conditions. Under the optimized conditions, a variety of aromatic, heteroaromatic, and alicyclic alcohols have been examined for their transformations to the corresponding carbonyl compounds. The mechanistic studies emphasize the important role of substrate (alcohol) and solvent (DMSO) in the generation of superoxide radical which is a vital intermediate for the transformation. This study also highlights the role of air as the oxidant in the oxidation process. Further, the practical application of the strategy has also been demonstrated for the oxidation of the alcoholic moiety in cholesterol.

Empowering Transition-Metal-Free Cascade Protocol for the Green Synthesis of Biaryls and Alkynes.

The work being presented in this paper demonstrates the simple and efficient "transition-metal-free" and "light-mediated" synthetic protocol for the synthesis of aryl iodides/biaryls/alkynes from aryl bromides. Under ultraviolet irradiation and in basic aqueous media, aryl bromides undergo transformation into aryl iodides which efficiently couple via a cascade reaction with a wide range of terminal alkynes/unactivated arenes to generate target molecules under green conditions.

Giant Placental Chorangioma: A Rare Case Report.

Chorangioma is a nontrophoblastic benign vascular tumour of the placenta, arising from the primitive chorionic mesenchyme. The clinical significance is related to the size of the tumours. Small chorangiomas, with a frequency of about 1%, are often asymptomatic. On the contrary, giant chorangiomas, greater than 5 cm in diameter, are rare tumours, with prevalence ranging from 1:9,000 to 1:50,000, and often associated with a variety of pregnancy complications and a poor perinatal outcome. We report a case of 26-year-old female who presented to us at 36 weeks of gestation with pain in the lower abdomen. Ultrasonograpy revealed polyhydramnios and a vascular tumour on the surface of placenta. Proper conservative antenatal management was done and a full term healthy baby was delievered. Histopathological examination of the extracted mass confirmed the diagnosis of chorangioma. The novelty of this report lies in the presence of large nontrophoblastic vascular placental tumour and the absence of any fetal complications. We emphasise the need of regular and timely antenatal management to diagnose and treat the complications of chorangioma at an early stage.

Mutational and Structural Analysis of Conserved Residues in Ribose-5-Phosphate Isomerase B from Leishmania donovani: Role in Substrate Recognition and Conformational Stability.

Ribose-5-phosphate isomerase B from Leishmania donovani (LdRpiB) is one of the potential drug targets against visceral leishmaniasis. In the present study, we have targeted several conserved amino acids for mutational analysis (i.e. Cys69, His11, His102, His138, Asp45, Tyr46, Pro47 and Glu149) to gain crucial insights into their role in substrate binding, catalysis and conformational stability of the enzyme. All the eight LdRpiB variants were cloned, sequenced, expressed and purified. C69S, H102N, D45N and E149A mutants exhibited complete loss of enzyme activity indicating that they are indispensable for the enzyme activity. Kinetic parameters were altered in case of H138N, H11N and P47A variants; however Y46F exhibited similar kinetic behaviour as wild type. All the mutants except H138N exhibited altered protein structure as determined by CD and fluorescence spectral analysis. This data was supported by the atomic level details of the conformational changes and substrate binding using molecular dynamic simulations. LdRpiB also exhibited activity with D-form of various aldose substrates in the order of D-ribose > D-talose > D-allose > D-arabinose. Our study provides insights for better understanding of substrate enzyme interactions which can rationalize the process of drug design against parasite RpiB.

Synchronous Lipoma arborescens of bilateral wrist: An extremely rare manifestation and a new perspective on etiopathogenesis.

Lipoma arborescens is a rare lesion wherein abnormal proliferation of adipocytes in the subsynovial region is seen. Whether it is neoplastic or not is still not entirely clear. Usually, Lipoma arborescens is seen unilaterally around knee joint but it can also be seen around tendon sheaths or bursa in adults. Multiple lesions are also reported. Involvement of wrist is extremely rare. We are presenting a case with bilateral wrist involvement by Lipoma arborescens which to the best of our knowledge is first one in English literature. There are a host of co-morbid/associated conditions reported with Lipoma arborescens out of which chronic irritation and inflammation appears to be most significant. We are proposing a novel perspective on etiopathogenesis of Lipoma arborescens based on extant understanding of cellular differentiation and information gleaned from fetal autopsies. The new perspective also explains the predilection for knee joint.

Lymphangiomatosis: Two cases with unique presentations, salience of nomenclature, and diagnosis.

We are presenting two unique cases of lymphangiomatosis without visceral and bony involvement and critically discussing the nomenclature used in the extant literature. The first case was a 12-year-old boy with ill-defined mass on the right cheek extending into the ipsilateral orbit leading to conjunctival lesion. The second case was of a 14-week-old infant showing diffuse swelling on nape of the neck. In addition there were raised patches on dorsal aspects of bilateral hands and feet. The biopsies from all the lesions showed similar histopathological features consistent with lymphangiomatosis. We propose that the term lymphangiomatosis should be used only in cases with histological features of lymphangiomatosis. The term should not be used in cases of multiple lymphangiomas. We conclude that the lesions clinically diagnosed as lymphangioma may turn out to be lymphangiomatosis. Extensive lymphangiomatosis without visceral or bony involvement may lead to intrauterine death.

Mucinous tubular and spindle cell carcinoma of kidney: A case report of an unusual tumor.

Mucinous tubular and spindle cell carcinoma (MTSCC) is a rare and unusual variant of renal cell carcinoma (RCC). It is important to differentiate this from the other variants particularly papillary RCC since MTSCC is generally low-grade, has low malignant potential and favorable prognosis. We are reporting a 40-year-old female presenting with right flank pain and well-circumscribed renal mass confined to the lower pole. Tumor consisted of tubules and cords separated by pale mucinous material in some areas, whereas other areas showed dense cellularity without significant mucin. The tumor cells were cuboidal or spindle and did not show significant atypical features. The patient underwent nephrectomy and is asymptomatic clinically and radiologically after 2 years.

Aggregates of a hetero-oligophenylene derivative as reactors for the generation of palladium nanoparticles: a potential catalyst in the Sonogashira coupling reaction under aerial conditions.

The utilization of Pd nanoparticles stabilized by aggregates of hetero-oligophenylene derivative 3 as an excellent catalyst in a copper/amine free Sonogashira coupling reaction under aerial conditions at room temperature has been demonstrated.

Renal cell carcinoma, unclassified with unique features.

Renal cell carcinoma, unclassified constitute about 3-4% of all renal carcinomas. It essentially is a tumor where more than morphological variants or subtypes are seen in a single tumor. Usually there is a mixture of 2-3 different types. However, in this particular case there were at least 5 different types of morphological patterns in a single tumor including areas of so-called rhabdoid differentiation. The patient underwent nephrectomy and has been asymptomatic for the last 3.5. years. To the best of our knowledge, this is the first case of its own kind in the published literature.

Mianserin, an antidepressant kills Leishmania donovani by depleting ergosterol levels.

In the present study, we have investigated the antileishmanial potential of mianserin, an antidepressant. Mianserin was found to inhibit both the promastigote and amastigote forms of the parasite in a dose dependant manner. The IC50 values for promastigotes and amastigotes were 21 µM and 46 µM respectively. Interestingly, mianserin failed to inhibit THP-1 differentiated macrophages up to 100 µM concentration thus, exhibiting parasite selectivity. When mianserin was incubated with recombinant Leishmania donovani 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) enzyme, it exhibited an IC50 value of 19.8 µM. Inhibition kinetics revealed competitive mode of enzyme inhibition as the Km increased with no change in Vmax. Further structural investigation of enzyme-inhibitor interaction revealed quenching of HMGR tryptophan intrinsic fluorescence with a K(sv) value of 3.025±0.37 M(-1) and an apparent binding constant of 0.0954 mM. We further estimated ergosterol levels which is a major component of Leishmania cell membrane. It is synthesized by HMGR enzyme, the first rate limiting enzyme of the sterol biosynthetic pathway. Analysis of ergosterol levels by HPLC revealed ∼2.5-fold depletion in mianserin treated promastigotes with respect to untreated parasites. This data was further validated by exogenous supplementation of mianserin treated cells with ergosterol and cholesterol. Reversal of growth inhibition was observed only upon ergosterol addition though it was refractory to cholesterol supplementation. Overall, our results demonstrate the possibility of repositioning of an antidepressant for the treatment of Visceral Leishmaniasis.

Chemical- and thermal-induced unfolding of Leishmania donovani ribose-5-phosphate isomerase B: a single-tryptophan protein.

Ribose-5-phosphate isomerase B (RpiB), a crucial enzyme of pentose phosphate pathway, was proposed to be a potential drug target for visceral leishmaniasis. In this study, we have analyzed the biophysical properties of Leishmania donovani RpiB (LdRpiB) enzyme to gain insight into its unfolding pathway under various chemical and thermal denaturation conditions by using fluorescence and CD spectroscopy. LdRpiB inactivation precedes the structural transition at lower concentrations of both urea and guanidine hydrochloride (GdHCl). 8-Anilinonapthalene 1-sulfonic (ANS) binding experiments revealed the presence of molten globule intermediate at 1.5 M GdHCl and a nonnative intermediate state at 6-M urea concentration. Acrylamide quenching experiments further validated the above findings, as solvent accessibility of tryptophan residues increased with increase in GdHCl and urea concentration. The recombinant LdRpiB was completely unfolded at 6 M GdHCl, whereas the enzyme molecule was resistant to complete unfolding even at 8-M urea concentration. The GdHCl- and urea-mediated unfolding involves a three-state transition process. Thermal-induced denaturation revealed complete loss of enzyme activity at 65 °C with only 20 % secondary structure loss. The formation of the well-ordered ß-sheet structures of amyloid fibrils was observed after 55 °C which increased linearly till 85 °C as detected by thioflavin T dye. This study depicts the stability of the enzyme in the presence of chemical and thermal denaturants and stability-activity relationship of the enzyme. The presence of the intermediate states may have major implications in the way the enzyme binds to its natural ligand under various conditions. Also, the present study provides insights into the properties of intermediate entities of this important enzyme.

Antileishmanial compounds from Moringa oleifera Lam.

The antileishmanial activity of extracts and phytoconstituents of Moringa oleifera Lam. was investigated in vitro against promastigotes of Leishmania donavani. The 70% ethanolic extract of roots and the methanolic extract of leaves showed moderate inhibitory activity with IC50 values of 83.0 microg/ml and 47.5 microg/ml, respectively. Antileishmanial activity of the methanolic extract of leaves increased upon fractionation, as its ethyl acetate fraction was found to be more active with an IC50 value of 27.5 microg/ml. The most active antileishmanial compound niazinin, a thiocarbamate glycoside isolated from this fraction, showed an IC50 value of 5.25 microM. Results presented in this study indicate that extracts from M. oleifera may be developed as an adjuvant therapy for the treatment of leishmaniasis.

Ketanserin, an antidepressant, exerts its antileishmanial action via inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) enzyme of Leishmania donovani.

Leishmaniasis is one of the major health problems existing globally. The current chemotherapy for leishmaniasis presents several drawbacks like toxicity and increased resistance to existing drugs, and hence, there is a necessity to look out for the novel drug targets and new chemical entities. Current trend in drug discovery arena is the "repurposing" of old drugs for the treatment of diseases. In the present study, an antidepressant, ketanserin, was found lethal to both Leishmania donovani promastigotes and intracellular amastigotes with no apparent toxicity to the cells. Ketanserin killed promastigotes and amastigotes with an IC50 value of 37 µM and 28 µM respectively, in a dose-dependent manner. Ketanserin was found to inhibit L. donovani recombinant 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) enzyme with an IC50 value of 43 µM. Ketanserin treated promastigotes were exogenously supplemented with sterols like ergosterol and cholesterol to rescue cell death. Ergosterol could recover the inhibition partially, whereas cholesterol supplementation completely failed to rescue the inhibited parasites. Further, HMGR-overexpressing parasites were generated by transfecting Leishmania promastigotes with an episomal pspα hygroα-HMGR construct. Wild-type and HMGR overexpressors of L. donovani were used to study the effect and mode of action of this inhibitor. The HMGR overexpressors showed twofold resistance to ketanserin. These observations suggest that the lethal effect of ketanserin is due to inhibition of HMGR, the rate-limiting enzyme of the ergosterol biosynthetic pathway. Since targeting of the sterol biosynthetic pathway enzymes may be useful therapeutically, the present study may have implications in treatment of leishmaniasis.

Exploring Leishmania donovani 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) as a potential drug target by biochemical, biophysical and inhibition studies.

3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase (HMGR), an NADPH dependant enzyme catalyzes the synthesis of mevalonic acid from HMG-CoA required for isoprenoid biosynthesis. The HMGR gene from Leishmania donovani was cloned and expressed. Genome analysis of L. donovani revealed that HMGR gene having an open reading frame of 1305 bp encodes a putative protein of 434 amino acids. LdHMGR showed optimal activity at pH 7.2 and temperature 37 °C. Kinetic analysis of this enzyme revealed Km values of 35.7 ± 2.5 µM for (R,S)-HMG-CoA and 70 ± 7.9 µM for the cofactor NADPH. On tryptophan fluorescence quenching, the Stern Volmer constant (Ksv), binding constant (Ka) and protein:cofactor stoichiometry for interaction of NADPH cofactor with the enzyme were found to be 6.0 ± 0.7 M(-1), 0.17 µM and 0.72 respectively. Polyclonal anti-rat HMGR antibody detected a band of ∼45 kDa in all phases of promastigote growth. Biophysical analysis of the secondary structure of LdHMGR confirmed the presence of 25.7 ± 0.35% alpha helicity. Thermal denaturation studies showed extreme stability of the enzyme with 60% helical structure retained at 90 °C. Statins (simvastatin and atorvastatin) and non-statin (resveratrol) effectively inhibited the growth of L. donovani promastigotes as well as the catalytic activity of the recombinant LdHMGR. Atorvastatin was found to be most potent antileishmanial inhibitor with an IC50 value of 19.4 ± 3.07 µM and a very lower concentration of 315.5 ± 2.1 nM was enough to cause 50% recombinant LdHMGR enzyme inhibition suggesting direct interaction with the rate limiting enzyme of the ergosterol biosynthetic pathway. Exogenous supplementation of ergosterol in case of atorvastatin and resveratrol treated cells caused complete reversal of growth inhibition whereas simvastatin was found to be ergosterol refractory. Cholesterol supplementation however, failed to overcome growth inhibition in all the cases. Overall our study emphasizes on exploring LdHMGR as a potential drug target for the development of novel antileishmanial agents.

An electrospray ms-coupled microfluidic device for sub-second hydrogen/deuterium exchange pulse-labelling reveals allosteric effects in enzyme inhibition.

In this work, we introduce an integrated, electrospray mass spectrometry-coupled microfluidic chip that supports the complete workflow for 'bottom up' hydrogen/deuterium exchange (HDX) pulse labelling experiments. HDX pulse labelling is used to measure structural changes in proteins that occur after the initiation of a reaction, most commonly folding. In the present case, we demonstrate the device on the ß-lactamase enzyme TEM-1, identifying active site changes that occur upon acylation by a covalent inhibitor and subtle changes in conformational dynamics that occur away from the active site over a period of several second after the inhibitor is bound. Our results demonstrate the power of microfluidics-enabled sub-second HDX pulse labelling as a tool for studying allostery and show some intriguing correlations with mutagenesis studies.

Identification and characterization of a novel Ribose 5-phosphate isomerase B from Leishmania donovani.

Leishmaniasis is a group of tropical diseases caused by protozoan parasites of the genus Leishmania. Due to the emergence of resistance to the available antileishmanial drugs there is an immediate need to identify molecular targets on which to base future treatment strategies. Ribose 5-phosphate isomerase (Rpi; EC 5.3.1.6) is a key enzyme of the pentose phosphate pathway (PPP) which catalyses the reversible aldose-ketose isomerization between Ribose 5-phosphate (R5P) and Ribulose 5-phosphate (Ru5P). It exists in two isoforms A and B. These two are completely unrelated enzymes catalyzing the same reaction. Analysis of the Leishmania infantum genome revealed that though the RpiB gene is present, RpiA homologs are completely absent. An absence of RpiBs in the genomes of higher animals makes this enzyme a possible target for the chemotherapy of Leishmaniasis. In this paper, we report for the first time the presence of B isoform of the Rpi enzyme in Leishmania donovani (LdRpiB) by cloning and molecular characterization of the enzyme. An amplified L. donovani RpiB gene is 519 bp and encodes for a putative 172 amino acid protein with a molecular mass of ∼19 kDa. An ∼19 kDa protein with poly-His tag at the C-terminal end was obtained by heterologous expression of LdRpiB in Escherichia coli. The recombinant form of RpiB was obtained in soluble and active form. The LdRpiB exists as a dimer of dimers i.e. the tetramer form. The polyclonal antibody against Trypanosoma cruzi RpiB could detect a band of ∼19 kDa with the purified recombinant RpiB as well as native RpiB from the L. donovani promastigotes. Recombinant RpiB obeys the classical Michaelis-Menten kinetics utilizing R5P as the substrate with a K(m) value of 2.4±0.6 mM and K(cat) value of 30±5.2 s(-1). Our study confirms the presence of Ribose 5-phosphate isomerase B in L. donovani and provides functional characterization of RpiB for further validating it as a potential drug target.