Establishment and function of chromatin organization at replication origins.

The origin recognition complex (ORC) is essential for initiation of eukaryotic chromosome replication as it loads the replicative helicase-the minichromosome maintenance (MCM) complex-at replication origins1. Replication origins display a stereotypic nucleosome organization with nucleosome depletion at ORC-binding sites and flanking arrays of regularly spaced nucleosomes2-4. However, how this nucleosome organization is established and whether this organization is required for replication remain unknown. Here, using genome-scale biochemical reconstitution with approximately 300 replication origins, we screened 17 purified chromatin factors from budding yeast and found that the ORC established nucleosome depletion over replication origins and flanking nucleosome arrays by orchestrating the chromatin remodellers INO80, ISW1a, ISW2 and Chd1. The functional importance of the nucleosome-organizing activity of the ORC was demonstrated by orc1 mutations that maintained classical MCM-loader activity but abrogated the array-generation activity of ORC. These mutations impaired replication through chromatin in vitro and were lethal in vivo. Our results establish that ORC, in addition to its canonical role as the MCM loader, has a second crucial function as a master regulator of nucleosome organization at the replication origin, a crucial prerequisite for efficient chromosome replication.

A VPS15-like kinase regulates apicoplast biogenesis and autophagy by promoting PI3P generation in Toxoplasma gondii.

Phosphoinositides are important second messengers that regulate key cellular processes in eukaryotes. While it is known that a single phosphoinositol-3 kinase (PI3K) catalyses the formation of 3'-phosphorylated phosphoinositides (PIPs) in apicomplexan parasites like Plasmodium and Toxoplasma, how its activity and PI3P formation is regulated has remained unknown. Present studies involving a unique Vps15 like protein (TgVPS15) in Toxoplasma gondii provides insight into the regulation of phosphatidyl-3-phosphate (PI3P) generation and unravels a novel pathway that regulates parasite development. Detailed investigations suggested that TgVPS15 regulates PI3P formation in Toxoplasma gondii, which is important for the inheritance of the apicoplast-a plastid like organelle present in most apicomplexans and parasite replication. Interestingly, TgVPS15 also regulates autophagy in T. gondii under nutrient-limiting conditions as it promotes autophagosome formation. For both these processes, TgVPS15 uses PI3P-binding protein TgATG18 and regulates trafficking and conjugation of TgATG8 to the apicoplast and autophagosomes, which is important for biogenesis of these organelles. TgVPS15 has a protein kinase domain but lacks several key residues conserved in conventional protein kinases. Interestingly, two critical residues in its active site are important for PI3P formation and parasitic functions of this kinase. Collectively, these studies unravel a signalling cascade involving TgVPS15, a novel effector of PI3-kinase in T. gondii and possibly other Apicomplexa, that regulate critical processes like apicoplast biogenesis and autophagy.

PI4-kinase and PfCDPK7 signaling regulate phospholipid biosynthesis in Plasmodium falciparum.

PfCDPK7 is an atypical member of the calcium-dependent protein kinase (CDPK) family and is crucial for the development of Plasmodium falciparum. However, the mechanisms whereby PfCDPK7 regulates parasite development remain unknown. Here, we perform quantitative phosphoproteomics and phospholipid analysis and find that PfCDPK7 promotes phosphatidylcholine (PC) synthesis by regulating two key enzymes involved in PC synthesis, phosphoethanolamine-N-methyltransferase (PMT) and ethanolamine kinase (EK). In the absence of PfCDPK7, both enzymes are hypophosphorylated and PMT is degraded. We further find that PfCDPK7 interacts with 4'-phosphorylated phosphoinositides (PIPs) generated by PI4-kinase. Inhibition of PI4K activity disrupts the vesicular localization PfCDPK7. P. falciparum PI4-kinase, PfPI4K is a prominent drug target and one of its inhibitors, MMV39048, has reached Phase I clinical trials. Using this inhibitor, we demonstrate that PfPI4K controls phospholipid biosynthesis and may act in part by regulating PfCDPK7 localization and activity. These studies not only unravel a signaling pathway involving PfPI4K/4'-PIPs and PfCDPK7 but also provide novel insights into the mechanism of action of a promising series of candidate anti-malarial drugs.

Protein kinase TgCDPK7 regulates vesicular trafficking and phospholipid synthesis in Toxoplasma gondii.

Apicomplexan parasites are causative agents of major human diseases. Calcium Dependent Protein Kinases (CDPKs) are crucial components for the intracellular development of apicomplexan parasites and are thus considered attractive drug targets. CDPK7 is an atypical member of this family, which initial characterization suggested to be critical for intracellular development of both Apicomplexa Plasmodium falciparum and Toxoplasma gondii. However, the mechanisms via which it regulates parasite replication have remained unknown. We performed quantitative phosphoproteomics of T. gondii lacking TgCDPK7 to identify its parasitic targets. Our analysis lead to the identification of several putative TgCDPK7 substrates implicated in critical processes like phospholipid (PL) synthesis and vesicular trafficking. Strikingly, phosphorylation of TgRab11a via TgCDPK7 was critical for parasite intracellular development and protein trafficking. Lipidomic analysis combined with biochemical and cellular studies confirmed that TgCDPK7 regulates phosphatidylethanolamine (PE) levels in T. gondii. These studies provide novel insights into the regulation of these processes that are critical for parasite development by TgCDPK7.

Identification of Mobility-Resolved N-Glycan Isomers.

Glycan analysis has evolved considerably during the last decade. The advent of high-resolution ion-mobility spectrometry has enabled the separation of isomers with only the slightest of structural differences. However, the ability to separate such species raises the problem of identifying all the mobility-resolved peaks that are observed, especially when analytical standards are not available. In this work, we report an approach based on the combination of IMSn with cryogenic vibrational spectroscopy to identify N-glycan reducing-end anomers. By identifying the reducing-end α and ß anomers of diacetyl-chitobiose, which is a disaccharide that forms part of the common core of all N-glycans, we are able to assign mobility peaks to reducing anomers of a selection of N-glycans of different sizes, starting from trisaccharides such as Man-1 up to glycans containing nine monosaccharide units, such as G2. By building an infrared fingerprint database of the identified N-glycans, our approach allows unambiguous identification of mobility peaks corresponding to reducing-end anomers and distinguishes them from positional isomers that might be present in a complex mixture.

Identification of N-glycan positional isomers by combining IMS and vibrational fingerprinting of structurally determinant CID fragments.

While glycans are present on the surface of cells in all living organisms and play key roles in most biological processes, their isomeric complexity makes their structural characterization challenging. Of particular importance are positional isomers, for which analytical standards are difficult to obtain. We combine ultrahigh-resolution ion-mobility spectrometry with collision-induced dissociation and cryogenic infrared spectroscopy to determine the structure of N-glycan positional isomers. This approach is based on first separating the parent molecules by SLIM-based IMS, producing diagnostic fragments specific to each positional isomer, separating the fragments by IMS, and identifying them by comparing their IR fingerprints to a previously recorded spectral database. We demonstrate this strategy using a bottom-up scheme to identify the positional isomers of the N-linked glycan G0-N, in which a terminal N-acetylglucosamine (GlcNAc) is attached to either the α-3 or α-6 branch of the common N-glycan pentasaccharide core. We then use IR fingerprints of these newly identified isomers to identify the positional isomers of G1 and G1F, which are biantennary complex-type N-glycans with a terminal galactose attached to either the α-3 or α-6 branch, and in the case of G1F a fucose attached to the reducing-end GlcNAc. Starting with just a few analytical standards, this fragment-based spectroscopy method allows us to develop a database which we can use to identify positional isomers. The generalization of this approach would greatly facilitate glycan analysis.

A new approach for identifying positional isomers of glycans cleaved from monoclonal antibodies.

Glycosylation patterns in monoclonal antibodies (mAbs) can vary significantly between different host cell types, and these differences may affect mAbs safety, efficacy, and immunogenicity. Recent studies have demonstrated that glycan isomers with the terminal galactose position on either the Man α1-3 arm or the Man α1-6 arm have an impact on the effector functions and dynamic structure of mAbs. The development of a robust method to distinguish positional isomers of glycans is thus critical to guarantee mAb quality. In this work, we apply high-resolution ion mobility combined with cryogenic infrared spectroscopy to distinguish isomeric glycans with different terminal galactose positions, using G1F as an example. Selective enzymatic synthesis of the G1(α1-6)F isomer allows us to assign the peaks in the arrival-time distributions and the infrared spectra to their respective isomeric forms. Moreover, we demonstrate the impact of the host cell line (CHO and HEK-293) on the IgG G1F gycan profile at the isomer level. This work illustrates the potential of our approach for glycan analysis of mAbs.

Global Hunger Index does not really measure hunger - An Indian perspective.

The Global Hunger Index (GHI) is calculated and disseminated annually. India, which is the 5th largest economy in the world and has a good ranking in many other indicators, has a poor ranking based on this index. After a critical review of the appropriateness of the indicators used in GHI, the Indian Council of Medical Research has the viewpoint that the indicators of undernourishment, stunting, wasting and child mortality do not measure hunger per se. Referring to this index as a Hunger Index, and thereby ranking countries is not appropriate, since many of the measures that are used to evolve an index that measures hunger are probably contextual. Countries should therefore evolve their own measures that are suitable for their own context.

Using SLIM-Based IMS-IMS Together with Cryogenic Infrared Spectroscopy for Glycan Analysis.

The isomeric heterogeneity of glycans poses a great challenge for their analysis. While combining ion mobility spectrometry (IMS) with tandem mass spectrometry is a powerful means for identifying and characterizing glycans, it has difficulty distinguishing the subtlest differences between isomers. Cryogenic infrared spectroscopy provides an additional dimension for glycan identification that is extremely sensitive to their structure. Our approach to glycan analysis combines ultrahigh-resolution IMS-IMS using structures for lossless ion manipulation (SLIM) with cryogenic infrared spectroscopy. We present here the design of a SLIM board containing a series of on-board traps in which we perform collision-induced dissociation (CID) at pressures in the millibar range. We characterize the on-board CID process by comparing the fragments generated from a pentapeptide to those obtained on a commercial tandem mass spectrometer. We then apply our new technique to study the mobility and vibrational spectra of CID fragments from two human milk oligosaccharides. Comparison of both the fragment drift times and IR spectra with those of suitable reference compounds allows us to identify their specific isomeric form, including the anomericity of the glycosidic linkage, demonstrating the power of this tool for glycan analysis.

Analyzing glycans cleaved from a biotherapeutic protein using ultrahigh-resolution ion mobility spectrometry together with cryogenic ion spectroscopy.

Glycans covalently attached to protein biotherapeutics have a significant impact on their biological activity, clearance, and safety. As a result, glycosylation is categorized as a critical quality attribute that needs an adequate analytical approach to guarantee product quality. However, the isomeric complexity and branched structure of glycans makes their analysis a significant challenge. In this work, we propose a multidimensional approach for monitoring released glycans that combines ultrahigh-resolution ion mobility spectrometry (IMS) and cryogenic vibrational spectroscopy, and we demonstrate this technique by characterizing four N-glycans cleaved from the therapeutic fusion protein etanercept that range in abundance from 1% to 22% of the total N-glycan content. The recorded vibrational spectra exhibit well-resolved transitions that can be used as a fingerprint to identify a particular glycan. This work represents an important advance in the analysis of N-linked glycans cleaved from biopharmaceutical proteins that could eventually be used as tool for monitoring biopharmaceutical glycoforms.

Left Lateral Port: Safe Laparoscopic Port Entry in Previous Large Upper Abdomen Laparotomy Scar.

The first port entry in patient who underwent previous abdominal surgery. Palmer's point can be used in patients with suspected periumbilical adhesions, a history of an umbilical hernia, or multiple failed attempts of insufflations at the umbilicus. Palmer's point has its limitations in cases of left upper quadrant surgery, splenomegaly, portal hypertension, and improper nasogastric tube placement giving rise to a bloated stomach. In such cases, a new and safe point for laparoscopic entry is needed. In the present case of a patient who underwent previous upper abdominal surgery with the chevron incision obscuring Palmer's point, laparoscopic entry was made through a novel point that was found to be safe in such cases and can be used in similar cases of previously scarred abdomens.

A review of selected nutrition & health surveys in India.

Assessment of the status of health and nutrition of a population is imperative to design and implement sound public health policies and programmes. The various extensive national health and nutrition surveys provide national-level information on different domains of health. These provide vital information and statistics for the country, and the data generated are used to identify the prevalence and risk factors for the diseases and health challenges faced by a country. This review describes the various national health and nutrition surveys conducted in India and also compares the information generated by each of these surveys. These include the National Family Health Survey, District Level Household Survey, Annual Health Survey, National Nutrition Monitoring Bureau Survey, Rapid Survey on Children and Comprehensive National Nutrition Survey.

Comparison of haemoglobin estimates using direct & indirect cyanmethaemoglobin methods.

BACKGROUND & OBJECTIVES: Estimation of haemoglobin is the most widely used method to assess anaemia. Although direct cyanmethaemoglobin method is the recommended method for estimation of haemoglobin, but it may not be feasible under field conditions. Hence, the present study was undertaken to compare indirect cyanmethaemoglobin method against the conventional direct method for haemoglobin estimation. METHODS: Haemoglobin levels were estimated for 888 adolescent girls aged 11-18 yr residing in an urban slum in Delhi by both direct and indirect cyanmethaemoglobin methods, and the results were compared. RESULTS: The mean haemoglobin levels for 888 whole blood samples estimated by direct and indirect cyanmethaemoglobin method were 116.1 ± 12.7 and 110.5 ± 12.5 g/l, respectively, with a mean difference of 5.67 g/l (95% confidence interval: 5.45 to 5.90, P<0.001); which is equivalent to 0.567 g%. The prevalence of anaemia was reported as 59.6 and 78.2 per cent by direct and indirect methods, respectively. Sensitivity and specificity of indirect cyanmethaemoglobin method were 99.2 and 56.4 per cent, respectively. Using regression analysis, prediction equation was developed for indirect haemoglobin values. INTERPRETATION & CONCLUSIONS: The present findings revealed that indirect cyanmethaemoglobin method overestimated the prevalence of anaemia as compared to the direct method. However, if a correction factor is applied, indirect method could be successfully used for estimating true haemoglobin level. More studies should be undertaken to establish agreement and correction factor between direct and indirect cyanmethaemoglobin methods.

Deficiencies of Serum Ferritin and Vitamin B12, but not Folate, are Common in Adolescent Girls Residing in a Slum in Delhi.

Anemia among adolescent girls is one of the major challenges faced by India. The present study was undertaken to assess the prevalence of anemia and status of other hematological parameters among adolescent girls (11 - 18 years) residing in an urban slum of Delhi. A total of 794 adolescent girls were recruited for the study. The prevalence of anemia was estimated using the cyanmethemoglobin method. Serum levels of ferritin, folic acid and vitamin B12 were estimated for anemic subjects. The prevalence of anemia was reported as 58.7 %, with 31.6 %, 25.7 % and 1.4 % of subjects being mild, moderate and severely anemic. Hemoglobin levels of subjects who had attained menarche were found to be significantly lower than those who had not attained menarche. The prevalence of serum ferritin, folic acid and vitamin B12 deficiency among those who were anemic was reported as 41.1 %, 5.0 % and 63.3 % respectively. A total of 23.5 % anemic subjects had concomitant micronutrient deficiencies of serum vitamin B12 and ferritin. The results indicate that supplemental iron and vitamin B12 may better address the burden of anemia in adolescent girls in Delhi.

Effect of coconut and sesame oils on gingivitis.

The effectiveness of Oil Pulling Therapy (OPT) with coconut (CO) and sesame oil (SO) on gingivitis patients is of interest. Forty patients were randomly distributed into group A and B for CO and SO respectively. Participants of group A were explained in detail about the OPT with CO and group B with SO along with their routine oral hygiene practice for 30 days. The mean plaque index of CO and SO reduced from 1.5 to 1.32 and 1.65 to 1.36 (p<0.05) respectively after 30 days. The mean gingival index of CO and SO declined from 1.12 to 0.9 and 1.1 to 0.81 respectively after 30 days (p<0.05) compared to initial scores. The mean no. of colonies in the case of CO and SO declined from 35.8 x 103 to 32.4 x 103 and 6.8 x 103 to 34.6 x 103 after 30 days (p<0.05). OPT reduced plaque and gingivitis, according to the results of one month. Hence, we must increase awareness about oil pulling, as this home therapy can prevent gingival diseases in countries with limited resources like ours.

Mild Cognitive Impairment: Clinical Presentation and Short-Term Comparative Outcome of Patients With Potentially Modifiable and Nonmodifiable Risk Factors.

Objective: To estimate the prevalence and study the clinical presentation of mild cognitive impairment (MCI), assess its outcome in terms of cognition and quality of life, identify factors for reversion to baseline, and compare these factors in the modifiable and nonmodifiable risk factor groups.Methods: Individuals aged >50 years with memory/cognitive complaint(s) were screened using the Mini-Cog over 1 year (August 2018-August 2019). Those meeting the DSM-5 criteria for MCI were enrolled, and risk factors (modifiable and nonmodifiable) were noted. Assessments were done using the Hindi version of the Montreal Cognitive Assessment (H-MoCA), the Clinical Dementia Rating (CDR)-Hindi version, and the World Health Organization Quality of Life-Brief Hindi version. Treatment outcome was assessed at 6 months and compared between the risk factor groups. Factors for reversion of MCI were assessed.Results: A total of 124 patients (22.1% of 561 with cognitive complaints) had MCI, and 100 patients (50 patients from the modifiable group and 50 patients from the nonmodifiable group) completed the study. Depression (52%) and hypertension (48%) were common risk factors. End point cognition scores were similar in both groups, with quality of life better in the modifiable group (P = .023). Age was negatively correlated with cognition in total patients and the nonmodifiable group (r =0.283-0.420; P = .002-.004). In total patients, cognition moderately correlated with education and somewhat with quality of life; 31% and 57% reverted to normal on the MoCA and CDR scales, respectively, while 1 progressed to dementia. Reverters had higher baseline H-MoCA scores (odds ratio [OR] = 6.996; P < .001) and were treated with cholinesterase inhibitors + vitamin E (OR = 28.999; P = .007).Conclusion: Short-term outcome for both the modifiable and nonmodifiable risk factor groups was favorable. Higher education positively correlated with cognition, which itself predicted a better quality of life. Reverters of MCI had better baseline cognition and were treated with cholinesterase inhibitors + vitamin E.Prim Care Companion CNS Disord 2024;26(4):24m03708. Author affiliations are listed at the end of this article.

Comparison between Air Q and intubating laryngeal mask airway as intubation conduits in patients with simulated fixed cervical spine: a prospective observational study.

The intubating laryngeal mask airway (ILMA) can be used for ventilation and oxygenation between intubation attempts, but there is a varied success rate ranging from 33% to 96%. Air Q is a relatively new entrant. Parker flex tube aids in atraumatic intubation. The primary aim of this study was to compare Air Q intubating laryngeal airway with ILMA as intubation conduits in patients with simulated fixed cervical spine using a Parker flex tube. It was a single-blinded, randomized, prospective, and comparative study conducted on 91 patients aged between 18 to 60 years of either sex, scheduled to undergo elective surgery under general anesthesia belonging to the American Society of Anesthesiologists physical status I and II. Out of 45 patients in each group, Air Q was successfully placed in 43 patients and ILMA was successfully placed in 44 patients. 35.56% of the patients required maneuvers for placing the Air Q, whereas, for placing the ILMA, only 15.56% of the patients required maneuvers. Intubation through the AIR Q was successful in 39 patients and through the ILMA in 44 patients, but there was no significant difference between the two groups. The number of attempts and the time of device insertion were comparable. There were a similar number of attempts, maneuvers required, and time is taken for endotracheal intubation. The incidence of cough and sore throat was comparable in both groups. We conclude that ILMA has a higher success rate than Air Q for tracheal intubation with Parker Flex tube in patients with simulated fixed cervical spine. More optimized maneuvers were required for the placement of Air Q.

Multistage Ion Mobility Spectrometry Combined with Infrared Spectroscopy for Glycan Analysis.

The structural complexity of glycans makes their characterization challenging, not only because of the presence of various isomeric forms of the precursor molecule but also because the fragments can themselves be isomeric. We have recently developed an IMS-CID-IMS approach using structures for lossless ion manipulations (SLIM) combined with cryogenic infrared (IR) spectroscopy for glycan analysis. It allows mobility separation and collision-induced dissociation of a precursor glycan followed by mobility separation and IR spectroscopy of the fragments. While this approach holds great promise for glycan analysis, we often encounter fragments for which we have no standards to identify their spectroscopic fingerprint. In this work, we perform proof-of-principle experiments employing a multistage SLIM-based IMS-CID technique to generate second-generation fragments, followed by their mobility separation and spectroscopic interrogation. This approach provides detailed structural information about the first-generation fragments, including their anomeric form, which in turn can be used to identify the precursor glycan.

Drug Utilization Research and Predictors of Outcomes in the Intensive Care Unit of a Tertiary Care Hospital: A Prospective Observational Study.

BACKGROUND: Multiple drugs are commonly prescribed to intensive care unit (ICU) patients owing to the disease profile, multiple organ dysfunction, prophylaxis, management of stress ulcers, nosocomial infections, etc. This study aimed to evaluate the drug utilization patterns and factors influencing mortality and duration of stay in ICU patients.  Methodology: A prospective observational study was conducted in the ICU of our tertiary care hospital, Postgraduate Institute of Medical Sciences, Rohtak. Data was collected from treatment charts of patients using a structured pretested proforma. World Health Organization Anatomical Therapeutic Chemical/Defined Daily Dose (WHO ATC/DDD) methodology and core prescribing indicators were used to assess drug utilization data. The effect of different variables on mortality and duration of stay in the ICU was evaluated using regression analysis. RESULTS: An average of 8.78 drugs were prescribed per patient. Among the 922 prescriptions, anti-infectives, anti-inflammatory drugs, and drugs acting on the gastrointestinal tract were the most frequent medication classes prescribed. Polypharmacy and trade name prescribing were common. For most of the drugs, the prescribed daily dose corresponded to the WHO-DDD except ceftriaxone and levofloxacin. Age, presence of cardiac disorders, and Glasgow Coma Scale (GCS) score at admission directly correlated with mortality while the use of diuretics had a negative correlation with the duration of ICU stay.  Conclusions: There is a need to rationalize drug therapy in the ICU with regard to limiting polypharmacy and emphasizing generic drug name prescribing and adherence to the essential drug list. Antibiotic prescription patterns, in particular, deserve a special focus keeping in mind the multitude of factors demanding aggressive antibiotic use in critically ill intensive care patients.

Short-term Clinical Outcome of Previously Untreated and Treated Schizophrenia and Impact of Duration of Untreated Psychosis.

Background: Duration of untreated psychosis (DUP) is an important modifiable factor affecting schizophrenia outcomes. A dearth of research in India on untreated versus treated schizophrenia warrants further research. Methods: This was a longitudinal study in a tertiary hospital over 2 years. Inpatients diagnosed with schizophrenia (N = 116), aged 18-45, were divided into untreated and treated groups. Diagnostic confirmation, severity assessment, and clinical outcome were done using ICD-10 criteria, Positive and Negative Syndrome Scale (PANSS), and Clinical Global Impression (CGI) scale. Follow-up was done at 12 and 24 weeks. DUP was measured, and its association with the outcome was assessed. Results: Final analysis included 100 patients, 50 each of previously untreated and treated. Untreated patients had lower age and duration of illness (DOI), but higher DUP (p < .001). Treated patients showed much improvement on CGI-I at 12 weeks (p = .029), with no difference at 24 weeks. PANSS severity comparison showed no difference, and both groups followed a declining trend. In untreated patients, age of onset (AoO) was negatively correlated with severity (except general symptoms at baseline) at all follow-ups ('r' range = -0.32 to -0.49, p < .05), while DOI showed a positive correlation with negative and general symptoms at 12 weeks (r ~ 0.3, p < .05). Treated patients showed inconsistent and lower negative correlation between AoO and PANSS, with no correlation between severity and DOI. The mean sample DUP was 17.9 ± 31.6 weeks; it negatively correlated with education (r = -0.25, p = .01) and positively with PANSS severity ('r' range = 0.22 to 0.30, p < .05) at all follow-ups, especially negative symptoms. Patients with no or minimal improvement on CGI at 24 weeks had higher DUP (Quade's ANOVA F[1,98] = 6.24, p = .014). Conclusion: Illness variables in untreated schizophrenia affect severity, which has delayed improvement than treated schizophrenia. Higher DUP is associated with negative symptoms of schizophrenia.