Potential diagnostics and therapeutic approaches in COVID-19.

The most important aspect of controlling COVID-19 is its timely diagnosis. Molecular diagnostic tests target the detection of any of the following markers such as the specific region of the viral genome, certain enzyme, RNA-dependent RNA polymerase, the structural proteins such as surface spike glycoprotein, nucleocapsid protein, envelope protein, or membrane protein of SARS-CoV-2. This review highlights the underlying mechanisms, advancements, and clinical limitations for each of the diagnostic techniques authorized by the Food and Drug Administration (USA). Significance of diagnosis triaging, information on specimen collection, safety considerations while handling, transport, and storage of samples have been highlighted to make medical and research community more informed so that better clinical strategies are developed. We have discussed here the clinical manifestations and hospital outcomes along with the underlying mechanisms for several drugs administered to COVID-19 prophylaxis. In addition to favourable clinical outcomes, the challenges, and the future directions of management of COVOD-19 are highlighted. Having a comprehensive knowledge of the diagnostic approaches of SARS-CoV-2, and its pathogenesis will be of great value in designing a long-term strategy to tackle COVID-19.

Three-Dimensional Spinal Position With and Without Manual Distraction Load Increases Spinal Height.

OBJECTIVE: The purpose of this study was to investigate if spinal height increases using 3-dimensional (3-D) spinal position with and without manual distraction load and to assess the correlation between spine height changes and degrees of trunk rotation. METHODS: Fifty-six participants were randomly placed in one of two groups: (1) 3-D spinal position with manual distraction load, and (2) without manual distraction load. Spinal height was measured before and after the interventions using a stadiometer. For the statistical analysis, we used a 2 (Loading status: pre- versus post-intervention height) X 2 (3-D spinal position: with versus without manual distraction load) repeated measures Analysis of Variance (ANOVA) was used to identify significant interaction and main effects. Paired t-tests were used to calculate differences in spinal height changes between the two interventions. Pearson correlation coefficient was used to measure correlations between changes in spinal heights and degrees of trunk rotation. RESULTS: Mean spinal height increase with 3-D spinal position with and without manual distraction load was 6.30 mm (±6.22) and 5.69 mm (±4.13), respectively. No significant interaction effect was present between loading status and 3-D spinal position but a significant main effect in loading status was. Paired t-tests revealed significant differences in spinal heights between pre-and post-3-D spinal position with and without manual distraction load. No significant correlation was measured between trunk rotation and spinal height changes. CONCLUSION: 3-D spinal position with or without distraction load increased spinal height. This suggests that 3-D spinal positioning without manual distraction could be used in home settings to help maintain intervertebral disc (IVD) health.

Purified winged bean protease inhibitor affects the growth of Bactrocera cucurbitae.

The melon fruit fly, Bactrocera cucurbitae (Coquillett), is a serious agricultural pest which has defied the various control measures employed against it. Protease inhibitors present in plants which have the potential to inhibit the growth and development of associated insect pests can be a possible alternative which can be manipulated for developing resistance in plants to the pest. In the present study, winged bean (Psophocarpus tetragonolobus) protease inhibitor isolated through affinity chromatography was explored for its potential to disrupt the development of melon fruit fly, B. cucurbitae. Different concentrations (12.5, 25, 50, 100, 200, and 400 µg ml-1) of the winged bean protease inhibitor (WBPI) were incorporated into the artificial diet of the second instar (64-72 h old) larvae of B. cucurbitae. The WBPI significantly delayed the larval, pupal, and total development period. The percentage pupation and adult emergence of the treated larvae was reduced as compared with control. The activities of major digestive enzymes (trypsin, chymotrypsin, leucine aminopeptidase, and elastase) decreased significantly in the larvae treated with different concentrations (50, 100, 200, and 400 µg ml-1) of WBPI. The findings reveal that the inhibitor holds considerable promise for the management of the melon fruit fly.

A study of frequency distribution and some epidemiological features of reported cases and deaths of dengue in Ludhiana district during the year 2008.

There were 7337 cases and 50 deaths reported in the state of Punjab from 1997 to 2008 of which Ludhiana district of Punjab contributed 4973 cases and 32 deaths. Number of cases (2506) and deaths (13) reported were highest in Ludhiana in the year 2008. City/Area wise reporting of dengue cases in hospitals of Ludhiana in 2008 show that the disease is mostly prevalent in the urban areas but now has been reported from rural areas also as 2 cases have been reported from village Kumkalan, block Mangat, Ludhiana. Age wise distribution of the cases shows maximum incidence per lakh population in 31-50 years (52.06) followed by 16-30 years (49.00) and lowest in infants (0.36). The difference of incidence in various age groups has been found highly significant. Out of the 2988 cases reported from Ludhiana district, there were 1993 males, 989 females and no mention of the sex in 6 cases. The difference of incidence in males and females has been found highly significant. Month wise distribution of the cases shows maximum incidence per lakh population to be the highest in October (90.27) followed by November (33.39) and December (23.18). The difference of incidence in various months has been found highly significant. As the dengue is increasingly becoming an escalating problem it needs necessary control measures especially regarding BCC activities and appointment of Biologists at the district level in Punjab needs to be done.

Studies on the role of six enzymes in the metabolism of kinetin in mustard aphid, Lipaphis erysimi (Kalt.).

The activity of catalase, glutathione peroxidase, superoxide dismutase, O-demethylase, ATPase and succinate dehydrogenase, belonging to two main classes of detoxification enzymes (i.e. hydrolases and oxido-reductases), mostly involved in metabolism and degradation of xenobiotics in insects, were assessed under the influence of kinetin, a plant growth regulator (PGR). The nymphs (48-52 hr old) of Lipaphis erysimi (Kalt.) were permitted to feed on radish plant, Raphanus sativus L. treated with kinetin (400 ppm) for 13, 25 and 37 hr. It was found that the activity of catalase, glutathione peroxidase and superoxide dismutase increased significantly when compared with the control of the same age group, which indicated that these enzymes might be playing a significant role in the metabolism of kinetin in this insect. The activity of O-demethylase showed an increase up to 25 hr of the treatment but it decreased under prolonged treatment whereas the activity of succinate dehydrogenase fluctuated insignificantly. ATPase showed a decrease in the activity with the treatment suggesting kinetin's interference in synthesis of ATPase.

Evaluation of the possible role of five enzymes in the metabolism of IBA in mustard aphid, Lipaphis erysimi (Kalt.).

The enzymatic activity of five enzymes viz. Glutathione S-transferases, Esterases, NADH dehydrogenase, NADH oxidase and Glutathione reductase were assessed under the influence of Indole butyric acid (IBA) (400 ppm) in the nymphs (48-52h old) of mustard aphid, Lipaphis erysimi fed on radish plants treated for 13, 25 and 37h. The activity of Glutathione S-transferases, Esterases and NADH dehydrogenase increased compared to that found in the control of the same age group of nymphs and it was concluded that these enzymes might be involved in the metabolism of IBA. The other two enzymes, NADH oxidase and Glutathione reductase showed no significant increase in their activity compared to that in the control of the same age group. It was hypothesized that the latter enzymes do not play any significant role in the metabolism of IBA.

Evaluation of the pesticidal potential of the congress grass, Parthenium hysterophorus Linn. on the mustard aphid, Lipaphis erysimi (Kalt.).

Petroleum ether extracts of leaves, stem and inflorescence of Parthenium hysterophorus Linn. at 500, 1000, 2000 and 5000 ppm concentrations were tested in the laboratory for their toxic effects on the mean life span and progeny production of adults of the mustard aphid, Lipaphis erysimi (Kalt.). The investigations revealed a significant decrease in life span and progeny production with treatment. Among the three plant parts tested for their efficacy, the leaf extract showed the most significant effect in causing a dose dependent decline in both the life span and progeny production.

Synthesis and in vitro evaluation of a series of diketopiperazine inhibitors of plasminogen activator inhibitor-1.

We have synthesized and evaluated a series of diketopiperazine-based inhibitors of PAI-1. These studies resulted in the identification of 34 which inhibited PAI-1 in vitro with an IC(50)=0.2 microM. The synthesis and SAR of these compounds are described.

Influence of PGRs on carbohydrate content in Lipaphis erysimi (Kalt.).

Six compounds (IBA, chlorogenic acid, cytokinine, GA3, alar B-9 and maleic hydrazide) belonging to four different categories of plant growth hormones were used to study their effect on carbohydrate content in L. erysimi. The second instar nymphs (48 hr old) were given both dipping and leaf surface treatment with 1024 ppm concentration of compounds for two time intervals i.e. 48 and 96 hr. The carbohydrate content decreased after treatment with 4 of the plant growth regulators i.e. GA3, alar B-9, IBA and chlorogenic acid with maximum suppression in GA3 treatment. Cytokinine did not induce any derogatory influence on carbohydrate content. The treatment with maleic hydrazide, on the other hand enhanced the carbohydrate content. It could be concluded that the application of these PGRs affected the carbohydrate synthesis or metabolism.

Mucosal permeability after subclinical intestinal ischemia-reperfusion injury: an exploration of possible mechanisms.

Changes in mucosal permeability may be important in the etiology of necrotizing enterocolitis. The authors have previously shown that subclinical ischemia-reperfusion injury results in increased permeability in the rat intestine, and have partially characterized this phenomenon. In the present study the authors attempt to determine the mechanism by which these changes occur. Six-week-old rats underwent 10-minute superior mesenteric artery occlusion (SMAO) or sham, and mucosal permeability to 51CrEDTA was measured after 30 minutes. Rats were pretreated with saline, inhibitors of oxygen free radicals (superoxide dismutase+catalase, vitamin E, allopurinol, alpha-phenyl-N-tert butyl-nitrone), inhibitors of eicosanoids (indomethacin, quinacrine, diethylcarbamazine, 13-azaprostanoic acid), the putative cytoprotective agent prostaglandin E2, or the inhibitor of neutrophil free radical production fructose 1-6 diphosphate. None of the agents significantly attenuated the increase in mucosal permeability caused by SMAO, although indomethacin and prostaglandin E2 significantly exacerbated the permeability changes. To further explore the role of neutrophils, tissue myeloperoxidase was measured 30 minutes after SMAO. There was no significant difference in myeloperoxidase levels between sham and SMAO animals. These data suggest that the early increase in mucosal permeability after subclinical ischemia-reperfusion injury is not mediated by oxygen free radicals, eicosanoids, or neutrophils. The deleterious effect of indomethacin and prostaglandin E2 suggests a possible protective role for the cyclooxygenase system, but further studies are necessary to elucidate this possibility.

Mucosal permeability in the immature rat intestine: effects of ischemia-reperfusion, cold stress, hypoxia, and drugs.

Increased mucosal permeability may represent an important factor in the etiology of necrotizing enterocolitis (NEC). In the present study we used an immature rat model to assess the permeability effects of a number of stresses commonly seen in infants with NEC. In 10-day-old rats, mucosal permeability to 51Cr EDTA was measured after subjecting the animals to 10-minute ischemia-reperfusion injury, 30 minutes of hypoxia (14% oxygen), cold stress (4 degree C for 4 minutes), and intraperitoneal indomethacin (0.2 or 2.0 mg/kg) or theophylline (40 or 200 mg/kg). When compared with appropriate controls, mucosal permeability was found to be significantly increased by ischemia-reperfusion injury, hypoxia, and high-dose indomethacin, but not by cold, theophylline, or low-dose indomethacin. Renal clearance studies confirmed that elevated blood levels of 51Cr EDTA were due to increased permeability rather than decreased renal excretion of the probe. These studies confirm that mucosal permeability in the immature rat is increased by a variety of insults, and may represent a "common pathway" in the etiology of NEC.

Histamine does not mediate mucosal permeability changes after subclinical intestinal ischemia-reperfusion injury.

UNLABELLED: Subclinical intestinal ischemia-reperfusion injury (IRI) increases mucosal permeability, and may be an important mechanism in the etiology of necrotizing enterocolitis. The current study was designed to assess the role of histamine in mediating this phenomenon. Six-week-old rats underwent 10-minute superior mesenteric artery occlusion (SMAO) or sham operation, and ileal mucosal permeability to 51Cr EDTA was measured 30 minutes after reperfusion. Rats were pretreated with intravenous saline, mepyramine (6 mg/kg), or ranitidine (5 mg/kg). SMAO resulted in a significant increase in permeability compared to sham, which was not attenuated by either of the histamine antagonists. In a second experiment, mucosal permeability to 51Cr EDTA was measured in 6-week-old rats during aortic infusion of saline or histamine (0.5 mg/kg/min). There was no significant increase in permeability as a result of histamine infusion. In a third experiment, 6-week-old and 10-day-old rats underwent sham or 10-minute SMAO, and both portal vein and ileal tissue histamine levels were measured 30 minutes after reperfusion. There was no significant difference between sham and SMAO with respect to portal vein histamine or tissue histamine at either age. IN CONCLUSION: (1) increased permeability was not blocked by either H1 or H2 blockers; (2) histamine infusion did not increase permeability; and (3) SMAO did not increase portal vein or tissue histamine levels. These data suggest that histamine does not play a role in mediating the increase in permeability after subclinical IRI in this model.

Mucosal permeability to 51Cr EDTA following subclinical intestinal ischemia-reperfusion injury in the weanling rat.

The etiology of necrotizing enterocolitis (NEC) is uncertain. We have hypothesized that subclinical intestinal ischemia might result in increased mucosal permeability to intraluminal toxins or bacteria, resulting in inflammation and NEC. In order to pursue this hypothesis, we designed a series of studies to investigate whether the first assumption is correct, ie whether a subclinical ischemia-reperfusion injury (IRI) results in increased mucosal permeability. Using a model of superior mesenteric artery occlusion (SMAO) in weanling rats, we initially defined 10-minute SMAO as "subclinical" IRI (ie, 100% survival, no histological changes, and no hemodynamic instability). Mucosal permeability to a standard probe molecule (51Cr EDTA) was then measured after sham operation, or 2-minute or 10-minute SMAO. There was an early increase in permeability 30 minutes after reperfusion in the 10-minute SMAO group, which was completely reversed by 2 hours. Further studies suggested that having passed through the mucosa, the probe entered the systemic circulation via both portal venous and intestinal lymphatic routes. Subclinical intestinal IRI results in an early, reversible increase in mucosal permeability to 51Cr EDTA, which may be important in the pathogenesis of NEC. Further studies are required to fully characterize this phenomenon, and to determine the mechanisms by which it occurs.

Increased mucosal permeability after intestinal ischemia-reperfusion injury is mediated by local tissue factors.

Subclinical intestinal ischemia-reperfusion injury (IRI) causes an increase in mucosal permeability and may represent an early event in the pathogenesis of necrotizing enterocolitis. The present study was undertaken to determine whether these changes are mediated by local or systemic factors. In 6-week-old weanling rats, the ileum was divided into two isolated loops with separate vascular supplies. The mesentery of the proximal loop was occluded for 30 minutes, following which the bowel was reperfused; permeability to 51Cr EDTA was then assessed in the distal loop 30 minutes after reperfusion. In control groups, the distal loop was subjected to 30-minute IRI ("positive" control) or 30-minute sham operation ("negative" control). Permeability in the distal loop was increased only with IRI to the distal bowel (15.4 +/- 3 counts/min/standard), and not with IRI to the proximal bowel (5.1 +/- 1) or with sham operation (8.5 +/- 2). To determine whether a mild "priming" injury might be necessary for systemic factors to have an effect, the distal loop was subjected to 2-minute IRI and the proximal to 30-minute IRI or sham. Permeability was not increased in the distal loop in either of these groups (5.7 +/- 1 and 7.8 +/- 2, respectively). Thirty-minute IRI in the proximal loop did not increase permeability in the distal loop, with or without a priming injury. Only direct IRI in the distal loop resulted in a significant increase in permeability. We conclude that the permeability changes in this model are mediated through local tissue effects, rather than by systemic factors.

Use of electron beam irradiation for mold decontamination on cheddar cheese.

Cheddar cheese slices, surface-inoculated with either Penicillium cyclopium or Aspergillus ochraceus spores, were vacuum packaged and irradiated using an electron beam accelerator. Following treatment at .21 and .52 kGy, the shelf-life of cheese containing P. cyclopium was extended by 3 and 5.5 d, respectively, in comparison with inoculated, untreated samples. Under similar treatment and storage conditions, cheese containing A. ochraceus exhibited average shelf-life extensions of 42.5 and 52.2 d, respectively. Increasing the postirradiation storage temperature to 15 degrees C reduced the shelf-life of cheese, especially with samples containing A. ochraceus. The lowest dose required to inactivate ca. 50 to 60 spores/cm2 of either A. ochraceus or P. cyclopium on the surface of cheese was ca. .42 and .95 kGy, respectively. Irradiation survival curves of A. ochraceus and P. cyclopium spores in cheese yielded average values (the dose required to reduce initial population by 90%) of .21 and .42 kGy, respectively.

An unusual repetitive gene family in Theileria parva which is stage-specifically transcribed.

Tpr1 is a repetitive DNA element from Theileria parva which has previously been shown to be of value in strain characterisation. Further characterisation, described here, has shown that Tpr1 is present in long tandem arrays. The sequence of 8.1 kb from one end of an array has been determined. The sequence showed that Tpr1 is a 1.44-kb element which contains an ORF extending from its 5' end to the 3' end. The sequenced region contains 4 large ORFs; 2 of these consisted only of Tpr1 whilst the third consisted of Tpr1 and a 0.55-kb element (Tpr2) located 5' of Tpr1. The largest ORF consisted of Tpr1 plus Tpr2 as well as an additional 420-bp element (Tpr3) 5' of Tpr2, thus a continuous ORF arranged 5'-Tpr3-Tpr2-Tpr1-3' was formed. This ORF potentially encodes a 795 amino acid polypeptide commencing at an ATG close to the 5' end. In contrast the first in frame ATGs in the other 3 ORFs are at least 417 bp from the 5' end. Southern analysis confirmed that the sequenced region was typical of the rest of the Tpr array(s). Transcripts containing both Tpr3 and Tpr1 were detected in the piroplasm but not the schizont stages of the life cycle.

Improved characterization of Theileria parva isolates using the polymerase chain reaction and oligonucleotide probes.

Theileria parva DNA was purified from piroplasms isolated from cattle infected with 5 different East African isolates of the parasite, including the East Coast fever reference stock T. p. parva Muguga. Total cellular DNA was prepared from T. parva schizont-infected bovine lymphoblastoid cell cultures (11 isolates). Two probes, previously isolated from T. p. parva Muguga repetitive genomic DNA, were hybridized to restriction digests; closely similar restriction fragment length polymorphism (RFLP) patterns were produced, and no two isolates had the same RFLP pattern. The DNA sequences of probe PMB3, two further copies of the repeated sequence from T. p. parva Muguga, and homologous regions from T. p. parva Kiambu 4 and T. p. lawrencei 3081, were determined. Oligonucleotides were synthesized corresponding to two conserved sections flanking a region which varied between isolates. These oligonucleotides were used as primers in the polymerase chain reaction to amplify the variable region. Further oligonucleotides corresponding to sequences in this variable region were able to distinguish between isolates and no sample hybridized to both oligonucleotides. This is the first unequivocal plus/minus discrimination between groups of isolates to be achieved for T. parva.

Oxygenated fluorocarbon perfusion as treatment of acute spinal cord compression injury in dogs.

Experiments were conducted to determine the therapeutic value of subarachnoid perfusion of the traumatized dog spinal cord with the fluorocarbon, Fluosol-DA (20%). Control dogs without lesions, but which had durotomy, subarachnoid catheter placement, and saline irrigation for 4 hours, did not have any residual neurological deficit. A series of 41 dogs underwent an acute spinal cord compression using an epidural balloon inflated to a pressure of 160 mm Hg and maintained for 1 hour. Treatment included durotomy only (11 dogs), durotomy with saline perfusion at room temperature (15 dogs), and durotomy with oxygenated Fluosol-DA perfusion at room temperature (15 dogs). The dogs underwent daily grading of neurological status for a 60-day period. Dogs undergoing perfusion of the spinal cord with either saline or oxygenated Fluosol-DA had significantly improved motor recovery (p less than 0.004) compared with dogs undergoing durotomy only. Perfusion with oxygenated Fluosol-DA resulted in significantly better motor recovery (p less than 0.05) than did perfusion with normal saline. Microscopic examination of the traumatized spinal cords failed to reveal a substantial difference between the three groups. However, dogs with better functional results tended to have less destruction of the white matter. Hemorrhagic necrosis of the central gray matter was consistently observed in all traumatized spinal cords.