Audit of Clinical Care Received by COVID-19 Patients Treated at a Tertiary Care Hospital of Nepal in 2021.

Like the world over, Nepal was also hard hit by the second wave of COVID-19. We audited the clinical care provided to COVID-19 patients admitted from April to June 2021 in a tertiary care hospital of Nepal. This was a cohort study using routinely collected hospital data. There were 620 patients, and most (458, 74%) had severe illness. The majority (600, 97%) of the patients were eligible for admission as per national guidelines. Laboratory tests helping to predict the outcome of COVID-19, such as D-dimer and C-reactive protein, were missing in about 25% of patients. Nearly all (>95%) patients with severe disease received corticosteroids, anticoagulants and oxygen. The use of remdesivir was low (22%). About 70% of the patients received antibiotics. Hospital exit outcomes of most (>95%) patients with mild and moderate illness were favorable (alive and discharged). Among patients with severe illness, about 25% died and 4% were critically ill, needing further referral. This is the first study from Nepal to audit and document COVID-19 clinical care provision in a tertiary care hospital, thus filling the evidence gap in this area from resource-limited settings. Adherence to admission guidelines was excellent. Laboratory testing, access to essential drugs and data management needs to be improved.

A rhodamine based biocompatible chemosensor for Al3+, Cr3+ and Fe3+ ions: extraordinary fluorescence enhancement and a precursor for future chemosensors.

A rhodamine based chemosensor, 3-(((2-(3',6'-bis(ethylamino)-2',7'-dimethyl-3-oxospiro[isoindoline-1,9'-xanthen]-2-yl)ethyl)imino)methyl)-2-hydroxy-5-methylbenzaldehyde (HL-CHO), has been developed for the detection of Al3+, Cr3+ and Fe3+ ions. The absorbance of HL-CHO at 528 nm increases significantly in HEPES buffer in methanol : water (9 : 1, v/v) (pH 7.4) in the presence of Al3+, Cr3+ and Fe3+ ions with the alteration of solution color from colorless to pink. The fluorescence intensity of the probe at 550 nm enhances by 1465, 588 and 800 fold in the presence of Al3+, Cr3+ and Fe3+ ions, respectively. To the best of our knowledge, this huge increase in fluorescence intensity with Al3+ and Cr3+ has not been observed for other rhodamine based chemosensing systems. The weak fluorescence and no coloration of the probe are due to the existence of a spirolactam ring. The trivalent cations induce the opening of the spirolactam ring and consequently change the color and the fluorescence intensity followed by the 1 : 1 complex formation with HL-CHO which are evident from Job's analysis, ESI mass spectral analysis and elemental analysis. The quantum yield and lifetime of HL-CHO have increased considerably in the presence of the trivalent cations. The high sensitivity of the probe towards all the cations is evident from the nM order of LOD values. This has been used in living cell imaging studies with the human neuroblastoma SH-SY5Y cell line. Having appended -CHO groups for Schiff-base condensation with other amines, HL-CHO could be a potential precursor for future chemosensors.

A Lysosome-Targetable Fluorescence Sensor for Ultrasensitive Detection of Hg2+ in Living Cells and Real Samples.

A new lysosome-targetable fluorescence sensor, Lyso-HGP, was designed and synthesized based on 4-methyl-2,6-diformylphenol as a fluorophore. Lyso-HGP displays highly sensitive fluorescent detection of Hg2+ in HEPES buffer solution (10 mM, DMSO 1%) of pH 7.0 at 37 °C due to the formation of highly fluorescent formyl-functionalized derivative Lyso-HGP-CHO. The sensor triggered a "turn-on" fluorescence response to Hg2+ with a simultaneous increase of fluorescence intensity by 180-fold just after 10 min. The response is very selective over a variety of biologically relevant cations, anions, molecules, and competitive toxic heavy metal cations. The limit of detection (LOD) was calculated as low as 6.82 nM. So, it can be utilized to detect this toxic heavy metal in biology and environmental samples in an aqueous buffer medium. Also, the sensor is able to monitor the subcellular distribution of Hg2+ specifically localized in the lysosome's compartment in the MCF7 human breast cancer cell line by fluorescence microscopy.

Pyridoxal Based Fluorescent Chemosensor for Detection of Copper(II) in Solution With Moderate Selectivity and Live Cell Imaging.

A pyridoxal-based fluorescent probe HL was synthesized for the detection of Cu(2+) in methanol with moderate selectivity. Upon addition of Cu(2+), to the solution of the probe in methanol exhibited a remarkable change in emission at 500 nm. With the limit of detection of 10 µM, the probe could well meet the recommended (less than 32 µM in drinking water) of the World Health Organization (WHO). The intracellular Cu(2+) imaging behaviour of HL was carried out on HeLa cells.

A new fluorogenic probe for the selective detection of carbon monoxide in aqueous medium based on Pd(0) mediated reaction.

A coumarin-based fluorogenic probe, PCO-1, senses carbon monoxide (CO) selectively in HEPES buffer at pH 8.0 through the intramolecular cyclization-elimination pathway based on Pd(0) mediated reaction. The probe exhibits a 'turn-on' response of CO over a variety of relevant reactive oxygen, nitrogen and sulfur species.

A novel chromo- and fluorogenic dual sensor for Mg(2+) and Zn(2+) with cell imaging possibilities and DFT studies.

A diformyl-p-cresol (DFC)-8-aminoquinoline based dual signaling probe was found to exhibit colorimetric and fluorogenic properties on selective binding towards Mg(2+) and Zn(2+). Turn-on fluorescent enhancements (FE) as high as 40 fold and 53 fold in 9 : 1 MeCN/water (v/v) at pH 7.2 in HEPES buffer for Mg(2+) and Zn(2+), respectively, were observed. The binding constants determined from the fluorescence titration data are: K = (1.52 ± 0.21) × 10(5) M(-1) and (9.34 ± 4.0) × 10(3) M(-2) at n = 1 and 0.5, for Mg(2+) and Zn(2+), respectively. The L : M binding ratios were also determined by Job's method, which support the above findings. This is further substantiated by HRMS analysis. Due to solubility in mixed organo-aqueous solvents as well as cell permeability it could be used for the in vitro/in vivo cell imaging of Mg(2+) and Zn(2+) ions with no or negligible cytotoxicity. This probe could be made selective towards Mg(2+) over Zn(2+) in the presence of TPEN, both under intra- and extracellular conditions and is superior to other Mg(2+) probes which suffer from selectivity of Mg(2+) over Ca(2+) or Zn(2+). Furthermore the dissociation constant (Kd = 6.60 µM) of the Mg(2+)-() complex is far lower than the so far reported Mg(2+) probes which fall in the mM range.

Effect of substituents on FRET in rhodamine based chemosensors selective for Hg2+ ions.

The effect of substituents on FRET in two newly designed rhodamine-based Hg(2+) ion selective chemosensors (L¹ and L²) has been explored by a systematic experimental and theoretical study. Comparison of these sensors in the analytical study and imaging of Hg(2+) ions in living cells has also been included.

A novel copper(II) complex as a nitric oxide turn-on fluorosensor: intracellular applications and DFT calculation.

We report, herein, the development of an easily synthesizable novel dansyl-based turn-on NO sensor L2. The UV-Vis titration data of L2 with Cu(2+) display a gradual increase in absorbance at 418 nm with [Cu(2+)], which were analyzed by using a non-linear least-squares computer-fit program yielding K = (1.16 ± 0.36) × 10(6) M(-1) and n = (1.28 ± 0.03) indicating a 1 : 1 complexation. The ground state geometries of L2 as well as its complex [Cu(L2)Cl](+) (1) were optimized by DFT calculations which showed that in complex 1 the central metal ion is in distorted tetrahedral geometry with bond distances very close to those found in analogous Cu(2+) complexes. The fluorescence of L2 was dramatically quenched (∼60-fold) through complexation with paramagnetic Cu(2+) to form [Cu(L2)Cl](+) in MeCN-H2O (9 : 1, v/v) at pH 7.2 in HEPES buffer, which on further treatment with Angeli's salt (Na2N2O3) restores its fluorescence property by ∼15-fold due to the reduction of Cu(2+) to Cu(+) by NO generated in solution from Na2N2O3. The lifetime measurements displayed a substantial decrease in the lifetime of free ligand L2 (τ0 = 12 ns) on complexation with Cu(2+) (τ0 = 2.1 ns). The detection limit of NO calculated by the 3σ method gives a value of 1.6 nM. The NO induced fluorescence enhancement of [Cu(II)(L2)Cl](+) was due to the reduction of [Cu(II)(L2)Cl](+) (1) to [Cu(I)(L2)](+) (2) and is supported by the disappearance of the d-d transition band at 850 nm as well as the X-band EPR signal of 1. The selective "turn on" fluorogenic behavior of L2 was examined on HeLa cells of human cervical cancer origin by fluorescence microscopy which showed very intense intracellular fluorescence that was strongly suppressed by the addition of Cu(2+) but it regains its fluorescence property on further incubation with Angeli's salt (Na2N2O3). The existence of [Cu(II)(L2)Cl](+) and [Cu(I)(L2)](+) in solution was confirmed by ESI-MS(+) (m/z) analysis. The effect of different biologically relevant cations and anions on the fluorescence property of L2 indicates that it was only the [Cu(II)(L2)Cl](+) which displayed high selectivity for NO, indicating its suitability for intracellular application without much worry about its cytotoxicity in a specified dose.

A 7-nitrobenz-2-oxa-1,3-diazole based highly sensitive and selective turn-on chemosensor for copper(II) ion with intracellular application without cytotoxicity.

A 7-nitrobenz-2-oxa-1,3-diazole (NBD) derived turn-on fluorescent probe (1) exhibits a reversible binding to Cu(2+) ion in presence of other metal ions, giving ~20 fold increase in fluorescence intensity. The apparent association constant (K(a)) for Cu(2+) was found to be 2.62 × 10(4) M(-1). The intracellular Cu(2+) imaging behaviour of chemosensor 1 on HeLa cells studied by fluorescence microscopy revealed that after incubation with 1 cells exhibited intensive fluorescence when exogenous Cu(2+) was introduced into the cell. Thus this probe features the ready availability, high sensitivity and selectivity towards Cu(2+) in MeCN-H(2)O (1:1, v/v) with cell imaging possibilities with no or negligible cytotoxicity.

[6]-Gingerol induces caspase 3 dependent apoptosis and autophagy in cancer cells: drug-DNA interaction and expression of certain signal genes in HeLa cells.

[6]-Gingerol, a pharmacologically important bioactive component of ginger, has been reported to have anti-hyperglycemic, anti-cancer and anti-oxidative properties, but mechanisms through which these are achieved are largely unclear. The present study focuses on apoptosis and autophagy, two key events of anti-cancer activity, in HeLa cells treated with [6]-gingerol. The treated cells showed several morphological changes, including externalization of phosphatidyl serine, degradation of DNA and increase in TUNEL positivity. Furthermore, there was depolarization of mitochondrial membrane potential, providing evidence of mitochondria mediated apoptosis. The expression of caspase 3 and PARP was increased in cells exposed to [6]-gingerol. Circular dichroism study for testing drug-DNA interaction with both calf thymus and nuclear DNA as target revealed that the drug had potential to bind with the nuclear DNA and induce conformational changes of DNA. The over-expression of NFkß, AKT and Bcl2 genes in cancer cells was down-regulated by [6]-gingerol treatment. On the other hand the expression levels of TNFα, Bax and cytochrome c were enhanced in [6]-gingerol treated cells. Thus, overall results suggest that [6]-gingerol has potential to bind with DNA and induce cell death by autophagy and caspase 3 mediated apoptosis.

A new half-condensed Schiff base compound: highly selective and sensitive pH-responsive fluorescent sensor.

A new probe, 3-[(3-benzyloxypyridin-2-ylimino)methyl]-2-hydroxy-5-methylbenzaldehyde (1-H) behaves as a highly selective fluorescent pH sensor in a Britton-Robinson buffer at 25 °C. The pH titrations show a 250-fold increase in fluorescence intensity within the pH range of 4.2 to 8.3 with a pK(a) value of 6.63 which is valuable for studying many of the biological organelles.

Thujone-Rich Fraction of Thuja occidentalis Demonstrates Major Anti-Cancer Potentials: Evidences from In Vitro Studies on A375 Cells.

CRUDE ETHANOLIC EXTRACT OF THUJA OCCIDENTALIS (FAM: Cupressaceae) is used as homeopathic mother tincture (TOΦ) to treat various ailments, particularly moles and tumors, and also used in various other systems of traditional medicine. Anti-proliferative and apoptosis-inducing properties of TOΦ and the thujone-rich fraction (TRF) separated from it have been evaluated for their possible anti-cancer potentials in the malignant melanoma cell line A375. On initial trial by S-diphenyltetrazolium bromide assay, both TOΦ and TRF showed maximum cytotoxic effect on A375 cell line while the other three principal fractions separated by chromatography had negligible or no such effect, because of which only TRF was further characterized and subjected to certain other assays for determining its precise anti-proliferative and apoptotic potentials. TRF was reported to have a molecular formula of C(10)H(16)O with a molecular weight of 152. Exposure of TRF of Thuja occidentalis to A375 cells in vitro showed more cytotoxic, anti-proliferative and apoptotic effects as compared with TOΦ, but had minimal growth inhibitory responses when exposed to normal cells (peripheral blood mononuclear cell). Furthermore, both TOΦ and TRF also caused a significant decrease in cell viability, induced inter-nucleosomal DNA fragmentation, mitochondrial transmembrane potential collapse, increase in ROS generation, and release of cytochrome c and caspase-3 activation, all of which are closely related to the induction of apoptosis in A375 cells. Thus, TRF showed and matched all the anti-cancer responses of TOΦ and could be the main bio-active fraction. The use of TOΦ in traditional medicines against tumors has, therefore, a scientific basis.

A highly selective fluorescent chemosensor for zinc ion and imaging application in living cells.

A new 2,6-bis(5,6-dihydrobenzo[4,5]imidazo[1,2-c]quinazolin-6-yl)-4-methylphenol (1) serves as a highly selective and sensitive fluorescent probe for Zn(2+) in a HEPES buffer (50 mM, DMSO:water = 1:9 (v/v), pH = 7.2) at 25 °C. The increase in fluorescence in the presence of Zn(2+) is accounted for by the formation of dinuclear Zn(2+) complex [Zn(2)(C(35)H(25)N(6)O)(OH)(NO(3))(2)(H(2)O)] (2), characterized by X-ray crystallography. The fluorescence quantum yield of the chemosensor 1 is only 0.019, and it increases more than 12-fold (0.237) in the presence of 2 equiv of the zinc ion. Interestingly, the introduction of other metal ions causes the fluorescence intensity to be either unchanged or weakened. By incubation of cultured living cells (A375 and HT-29) with the chemosensor 1, intracellular Zn(2+) concentrations could be monitored through selective fluorescence chemosensing.

In vitro and in vivo studies demonstrate anticancer property of root extract of Polygala senega.

Polygala senega is extensively used in traditional systems of medicine against various lung diseases including cancer. In the present study we tested the anticancer potentials of ethanolic extract of roots of P. senega (generally used as a homeopathic drug) in a mammalian model, where mice, in vivo, were treated chronically with benzo[a] pyrene and in vitro where lung adenocarcinoma cell line (A549) were used. We deployed various parameters like cell viability assay, chromatin condensation studies with Hoechst 333258 staining, and maintained suitable controls. To understand the possible signal transduction pathways, expression of various signal proteins such as Aryl Hydrocarbon receptor (AhR), cytochrome P450 (CYP1A1), Bcl-2, proliferating cell nuclear antigen (PCNA), Bax and Caspase-3 was studied. Additionally, reverse transcriptase polymerase chain reaction analysis of AhR, p53, PCNA and ß-actin (housekeeping) genes was made. Immunohistochemical localization of PCNA proteins was also conducted in vivo. Feeding of root extract of P. senega to mice (at the rate of 50 mg/kg and 100 mg/kg bw) chronically treated with the carcinogen (50 mg/kg bw dissolved in olive oil) showed positive modulation in expression of signal proteins. Upregulation of apoptotic signals such as p53, Caspase-3 and Bax, and downregulation of AhR, cytochrome P450 (CYP1A1), Bcl-2 and PCNA were observed. Addition of root extract of Polygala Senega (at doses of 50 µg and 100 µg) into culture medium containing A549 cells induced recovery of decreased cell viability and increased chromatin fragmentation (apoptosis). Therefore, results of both in vivo and in vitro studies scientifically validate its potential use as an anticancer agent, particularly against lung cancer, and provide important information potentially helpful in drug designing.

Lycopodine from Lycopodium clavatum extract inhibits proliferation of HeLa cells through induction of apoptosis via caspase-3 activation.

Crude ethanolic extract of the plant Lycopodium clavatum has long been used in complementary and alternative medicine for treating various liver ailments and Alzheimer's disease. It has also been claimed to have potential anti-cancer properties in vivo in mice chronically fed liver carcinogens, p-dimethylamino azobenzene (initiator) and phenobarbital (promoter). Incidentally, crude ethanolic extract of Lycopodium clavatum is a mixture of some 201 alkaloids. In order to ascertain if any major fraction can be attributed to have pronounced anti-cancer effect, we examined this major fraction by eluting the crude extract in petroleum ether:ethyl aetate (17:3 vol/vol;) solvent and tried to understand its underlying mechanism. Studies on morphological changes, cell viability and cytotoxicity by microscopy and FACS, Western blot and immunofluorescence of Bcl-2, Bax, cytochrome c, caspase-3 were conducted. Lycopodine was found to induce chromatin condensation, inter-nucleosomal DNA fragmentation and enhanced cell population in sub-G1 region along with increase in reactive oxygen species generation and mitochondrial membrane potential depolarization, release of cytochrome c and activation of caspase-3 which are the events closely involved in apoptosis. An overall analysis of results showed that Lycopodine considerably inhibited growth of HeLa cells which indicates its potential use in chemotherapy.

In vitro studies demonstrate anticancer activity of an alkaloid of the plant Gelsemium sempervirens.

The chemical structure of the main fluorescenting compound in the ethanolic extract (mother tincture) of the American yellow jasmine, Gelsemium sempervirens, was determined by employing (1)H nuclear magnetic resonance (NMR), (13)C NMR, mass spectroscopy, high-performance liquid chromatography (HPLC), correlation spectroscopy (COSY), and Fourier transform infrared (FTIR) spectroscopy analyses. Spectrofluorometric analysis has been made of the mother tincture and its agitated serial dilutions (up to 12th potency) prepared according to a homeopathic procedure in which serial, agitated dilutions were made separately in glass and polypropylene containers. The succussions were made by employing three different modes: hand jerk, sonication, and vortexing. The chemical formula of scopoletin, the main fluorescent compound, was determined to be C(10)H(8)O(4) having a molecular weight of 192.17. Significant differences were noted between the remedies prepared in the two types of containers. Further, a comparison between any two methods of agitation revealed significant differences in fluorometric data of remedies at certain potency levels. The biological (anticancer) action of the crude extract, the alkaloid scopoletin, and 2C potency of Gelsemium sp were tested in vitro on the HeLa cell line through fluorescence microscopy, the 3(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, and fluorescent activated cell sorting (FACS). The role of nanoparticles presumably derived from the containers, their orientation, and their interaction with the starting substance during the dynamization process initiated by different modes of agitation could possibly be attributed to the differences noted in the fluorometric data of potencies prepared in the two types of containers and among the three different means of succussion tested.

A sphingolipid rich lipid fraction isolated from attenuated Leishmania donovani promastigote induces apoptosis in mouse and human melanoma cells in vitro.

Lipids, especially sphingolipids, are emerging as inducer of apoptosis in a wide range of immortal cells, potentiating their therapeutic application in cancer. In the present study, a sphingolipid rich lipid fraction (denoted here as ALL), isolated from an attenuated strain of Leishmania donovani promastigote, was tested for its tumoricidal activity taking melanoma, the dreaded form of skin cancer cells, as model. ALL was found to induce chromatin condensation, internucleosomal DNA fragmentation and phosphatidylserine externalization with enhanced cell population in sub-G1 region in both mouse and human melanoma systems, namely B16F10 and A375 respectively. These are the hallmarks of cells undergoing apoptosis. Further analysis demonstrated that ALL treated melanoma cells showed significant increase in ROS generation, mitochondrial membrane potential depolarization, release of cytochrome c, and caspase-3 activation, which are the events closely involved in apoptosis. These findings indicate that one or more bioactive sphingolipid(s)/ceramide(s) present in ALL could be the causative agent(s) for the induction of apoptosis in melanoma cells. Further studies are thus necessary to identify these specific bioactive sphingolipid(s)/ceramide(s) and to establish their mechanism of action, in order to explore their use as anticancer agents.