Wound healing activity of aqueous extract of Crotalaria verrucosa in Wistar albino rats

ObjectiveTo evaluate the wound healing effect of aqueous extract of Crotalaria verrucosa (C. verrucosa) in rats.MethodsThree wound models including incision, excision and dead space wounds were used in this study. The parameters studied were breaking strength in incision models, granulation tissue dry weight, breaking strength and hydroxyproline content in dead space wounds, percentage of wound contraction and period of epithelialization in excision wound model.ResultsTwo doses of the extract with and without dexamethasone showed significant increases in mean hydroxyproline, total protein content and dry weight of granulation tissue but it was higher with dose 800 mg/kg comparing with the control. The dexamethasone treated group showed a significant (P<0.001) reduction in the wound breaking strength when compared to control group in incision type of wound model. Coadministration of C. verrucosa with dexamethasone significantly (P<0.001) increased the breaking strength compared to the dexamethasone treated only group. In excision wound model, the percentage of the wound contraction was significantly (P<0.01) increased by two doses of test extract on all the days except the lower dose which exhibited only on 12 th, 16 th days of drug treatment and it also reversed the dexamethasone suppressed wound contraction. It significantly (P <0.001) reduced the time required for epithelialization and reversed the epithelialization delaying effect of dexamethasone (P<0.001).ConclusionsC. verrucosa was found to possess significant wound healing property. This was evident by decrease in the period of epithelialization, increase in the rate of wound contraction, skin breaking strength, and granulation tissue dry weight content. Hence C. verrucosa could be a good wound healing agent.     

Can discourse processing performance serve as an early marker of Alzheimer’s disease and mild cognitive impairment? A systematic review of text comprehension

A number of linguistic and cognitive deficits have been reported during the course of Alzheimer’s disease (AD) and its preceding stage of mild cognitive impairment (MCI), with some deficits appearing years before onset of clinical symptoms. It continues to be a critical task to identify tools that may serve as an early marker of pathology that are also reliably able to distinguish AD from normal ageing. Given the limited success of classic psychometric cognitive testing, a novel approach in assessment is warranted. A potentially sensitive assessment paradigm is discourse processing. The aim of this review was to synthesize original research studies investigating comprehension of discourse in AD and MCI, and to evaluate the potential of this paradigm as a promising avenue for further research. A literature search targeting studies with AD or MCI groups over 60 years of age was conducted in PubMed, Web of Science, and PsycINFO databases. Eight articles with good quality were included in the review. Six measures of discourse comprehension—naming latency, summary, lesson, main idea, proportion of inferential clauses, true/false questions—were identified. All eight studies reported significant deficits in discourse comprehension in AD and MCI groups on five of the six measures, when compared to cognitively healthy older adults. Mixed results were observed for associations with commonly used cognitive measures. Given the consistent findings for discourse comprehension measures across all studies, we strongly recommend further research on its early predictive potential, and discuss different avenues for research.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10433-021-00619-5.     

Adventitial adipogenic degeneration is an unidentified contributor to aortic wall weakening in the abdominal aortic aneurysm

Objective

The processes driving human abdominal aortic aneurysm (AAA) progression are not fully understood. Although antiinflammatory and proteolytic strategies effectively quench aneurysm progression in preclinical models, so far all clinical interventions failed. These observations hint at an incomplete understanding of the processes involved in AAA progression and rupture. Interestingly, strong clinical and molecular associations exist between popliteal artery aneurysms (PAAs) and AAAs; however, PAAs have an extremely low propensity to rupture. We thus reasoned that differences between these aneurysms may provide clues toward (auxiliary) processes involved in AAA-related wall debilitation. A better understanding of the pathophysiologic processes driving AAA growth can contribute to pharmaceutical treatments in the future.

Beneficial effects of add-on raloxifene in schizophrenia

The role of estrogens in schizophrenia has been proposed from the observation of schizophrenia occurring later and with symptom severity being lesser in women. Utility of estrogens in treatment of psychoses, though seen to be useful, comes with inherent risks of neoplasias, given its agonistic action on breast and endometrium. This risk can be overcome with use of selective estrogen receptor modulators, like raloxifene. Raloxifene has been used in schizophrenia, with improvement in symptoms and cognitive functions. We report the use of raloxifene as an adjunctive treatment, with risperidone, in treatment-resistant form of schizophrenia. The patient, a 29-year-old woman, over a 7-month follow-up period, showed significant improvement in socio-occupational functioning, with reduction in symptom severity.     

Hepatoprotective activity of Terminalia paniculata against paracetamol induced hepatocellular damage in Wistar albino rats

ObjectiveTo evaluate the hepatoprotective activity of Terminalia paniculata against paracetamol induced hepatic damage in rats.MethodsThe plant material was shade dried, powdered and extracted with ethanol. Liv 52 and silymarin were used as standard drugs and 2% gum acacia as a control (vehicle). Alteration in the levels of biochemical markers of hepatic damage like AST, ALT, ALP and lipid peroxides were tested, and phytochemical tests were also performed.ResultsParacetamol (2 g/kg) increased the serum levels of alanine aminotransfer (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and the lipid peroxides. Treatment of Liv 52, silymarin and ethanolic extract of Terminalia paniculata (200 mg/kg) altered levels of biochemical marker and showed significant hepatoprotective activity. Ethanolic extract revealed the presence of phenolic compound and flavanoids. Our findings suggested that ethanolic bark extract of Terminalia paniculata possessed hepatoprotective activity in a dose dependent manner.ConclusionsTerminalia paniculata possesses hepatoprotective activity. It could be an effective and promising preventive agent against PCT induced hepatotoxicity.     

Electric field-driven microfluidics for rapid CRISPR-based diagnostics and its application to detection of SARS-CoV-2

The rapid spread of COVID-19 across the world has revealed major gaps in our ability to respond to new virulent pathogens. Rapid, accurate, and easily configurable molecular diagnostic tests are imperative to prevent global spread of new diseases. CRISPR-based diagnostic approaches are proving to be useful as field-deployable solutions. In one basic form of this assay, the CRISPR–Cas12 enzyme complexes with a synthetic guide RNA (gRNA). This complex becomes activated only when it specifically binds to target DNA and cleaves it. The activated complex thereafter nonspecifically cleaves single-stranded DNA reporter probes labeled with a fluorophore−quencher pair. We discovered that electric field gradients can be used to control and accelerate this CRISPR assay by cofocusing Cas12–gRNA, reporters, and target within a microfluidic chip. We achieve an appropriate electric field gradient using a selective ionic focusing technique known as isotachophoresis (ITP) implemented on a microfluidic chip. Unlike previous CRISPR diagnostic assays, we also use ITP for automated purification of target RNA from raw nasopharyngeal swab samples. We here combine this ITP purification with loop-mediated isothermal amplification and the ITP-enhanced CRISPR assay to achieve detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA (from raw sample to result) in about 35 min for both contrived and clinical nasopharyngeal swab samples. This electric field control enables an alternate modality for a suite of microfluidic CRISPR-based diagnostic assays.

Infectious diseases such as COVID-19 are a persistent global threat. Early-stage screening and rapid identification of infected patients are important during pandemics to treat the infected and to control disease spread. The frontline diagnostic tool for COVID-19 has been RT-PCR, and protocols for this have been developed and published by the World Health Organization (WHO) (1) and the US Centers for Disease Control and Prevention (CDC) (2). While these tests are specific and sensitive, they are laborious and time consuming, and are designed for large, centralized diagnostic laboratories.CRISPR-based diagnostic methods, including diagnostic assays for the COVID-19 pandemic (3, 4), have sparked great interest due to their versatility, sensitivity, and specificity. CRISPR applications for infectious disease diagnostics take advantage of a nuclease-like “collateral cleavage” induced by Cas12 and Cas13 enzymes (5, 6). In such methods, RNA-guided CRISPR-associated proteins such as Cas12 and Cas13 can be programmed to detect specific DNA and RNA sequences, respectively, from pathogens with single base pair specificity. For CRISPR–Cas12-based diagnostics, the CRISPR complex between the Cas12 enzyme and the synthetic guide RNA (gRNA) first recognizes and specifically cleaves (known as cis-cleavage) target pathogen single-stranded DNA (ssDNA) and/or double-stranded DNA (dsDNA) in the sample. The gRNA is designed to have between 18 and 24 nucleotides complementary to the target DNA sequence. This molecular recognition modifies the Cas12-gRNA complex into its “activated” form, which thereafter indiscriminately cleaves ssDNA molecules including synthetic ssDNA reporter molecules with fluorophore−quencher pairs. The nuclease-like characteristic of Cas12a on ssDNA, known as trans-cleavage, is activated only in the presence of target ssDNA or dsDNA activator (5). Thus, recognition of target DNA by Cas12a results in an increase in fluorescence signal due to the trans-cleavage activity of Cas12a on reporter molecules, and this recognition feature makes CRISPR useful for diagnostic applications.Despite advantages, several factors have impeded automated CRISPR-based detection methods. For example, the CRISPR–Cas12a-based method for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) developed by Broughton et al. (3) in March 2020 required upfront nucleic acid extraction and sample purification with a traditional adsorption/desorption column for purification, a process which typically takes up to 1 h. Moreover, Broughton et al. (3) carried out CRISPR enzymatic reactions in Eppendorf tubes and explored both colorimetric (using a lateral flow strip) and fluorescence readouts for target detection. Such protocols are not easily amenable to automation, consume significant reagent volume [typical CRISPR transcleavage assays are carried out in 50 to 100 μL volumes (3, 4)], and require 1 h or longer to complete, starting from raw sample. The consumption of reagents is important. For example, Joung et al. (4) reported supply chain constraints in procuring RPA (recombinase polymerase amplification) reagents for a CRISPR–Cas13-based test which they initially developed for SARS-CoV-2 in February 2020. This limitation compelled them to redesign their assay to one based on CRISPR–Cas12b and loop-mediated isothermal amplification (LAMP) in May 2020.Microfluidics offers important alternate strategies to accelerate biochemical reactions (7), multiplex (8), and automate CRISPR diagnostics. We here develop an electric field-enhanced microfluidic method that is broadly applicable to the field of CRISPR diagnostics. To this end, we use an electrokinetic microfluidic technique called isotachophoresis (ITP). ITP uses a two-buffer system which consists of a high-mobility leading electrolyte (LE) and a low-mobility trailing electrolyte (TE) buffer. On application of an electric field, sample ions with effective mobilities bracketed by the LE and TE ions selectively focus within an order 10 μm zone at the LE-to-TE interface. This focusing can preconcentrate, purify, mix, and accelerate reactions among sample and reagents species. ITP has been used to rapidly extract nucleic acids from a range of biological samples such as urine (9), blood (10), and cell lysates (11), and to accelerate DNA and RNA hybridization reactions (12).For ITP applications involving purification and extraction of nucleic acids, a proper choice of LE and TE ensures that target species (here, DNA and RNA) focus and preconcentrate in ITP, while leaving behind impurities and inhibitors to downstream analyses (here, inhibitors can include proteins and small cations) (10, 11). See Rogacs et al. (11) for a detailed review on purification of nucleic acids using ITP. When ITP is applied to control homogeneous biochemical reactions, a good choice of LE and TE enables all reacting species to cofocus and preconcentrate in ITP. The simultaneous preconcentration of all reactants in ITP accelerates product formation. As an example, Bercovici et al. (12) used ITP to demonstrate 14,000-fold acceleration of DNA hybridization assays. See Eid and Santiago (7) for a comprehensive review on ITP-enhanced biochemical reactions, including both homogeneous and heterogenous reactions.In this work, we combine microfluidics and on-chip electric field control to achieve two critical steps. First, we use ITP to automatically extract nucleic acids from raw biological samples, here, nasopharyngeal (NP) swab samples from COVID-19 patients and healthy controls. Second, we use electric field gradients in ITP to control and effect rapid CRISPR–Cas12 enzymatic activity upon target nucleic acid recognition. The latter is achieved using a tailored on-chip ITP process to cofocus Cas12–gRNA, reporter ssDNA, and target nucleic acids (SI Appendix, Figs. S1 and S2). This creates simultaneous mixing, preconcentration, and acceleration of enzymatic reactions. Our microfluidic method consumes minimal volume of reagents (order 100-fold lower than conventional methods) on-chip for CRISPR reactions and is amenable to automation. We apply our method to detection of SARS-CoV-2 RNA in an assay which takes around 30 min to 40 min from raw sample to result. We demonstrate this on clinical samples, including SARS-CoV-2 positive and negative clinical specimens. The method is both an alternate modality for CRISPR diagnostics and, to our knowledge, the fastest CRISPR-based detection of SARS-CoV-2 from raw samples with clinically relevant specificity and sensitivity.     

Central pressor actions of aminopeptidase-resistant angiotensin II analogs: challenging the angiotensin III hypothesis

Intracerebroventricular administration of angiotensins causes pronounced pressor and dipsogenic responses. The suggestion that angiotensin III rather than angiotensin II is the active peptide in the brain spawned what we call The Angiotensin III. HYPOTHESIS: To test this hypothesis, 5 angiotensin II analogs containing zero or one position substitutions conferring resistance to aminopeptidases were administered intracerebroventricularly to determine their pressor and dipsogenic efficacies. Two aminopeptidase-resistant analogs caused significantly greater pressor responses than angiotensin II, whereas 3 analogs caused pressor responses similar to angiotensin II. Latency to cause a pressor response for 4 of the 5 aminopeptidase-resistant angiotensin II analogs was the same as for angiotensin II. There was no detectable formation of (125)I-angiotensin III from 1 of the intracerebroventricularly administered analogs, (125)I- N-Methyl-l-Asp(1)-angiotensin II, indicating its aminopeptidase resistance. Latency to drink also did not differ between the angiotensins. After the initial dipsogenic response, water was removed until 25 minutes after angiotensin administration to avoid interfering with the pressor response. The dipsogenic stimulus was sustained 25 minutes after intracerebroventricular injection of angiotensin II and its aminopeptidase-resistant analogs. Comparison of angiotensin III and angiotensin II showed equivalent pressor responses with similar latencies and durations. The latency to drink was similar for angiotensin III and angiotensin II. However, there was no dipsogenic response to angiotensin III 25 minutes after intracerebroventricular injection. These data do not support The Angiotensin III Hypothesis and suggest that conversion of exogenously applied angiotensin II to angiotensin III is not necessary to cause brain-mediated pressor or dipsogenic responses.