Statewide Implementation of Virtual Perinatal Home Visiting During COVID-19.

PURPOSE: This evaluation describes efforts taken by MIECHV administrators and staff during the pandemic using data collected from 60 MIECHV staff surveys and nine statewide weekly focus groups. DESCRIPTION: The Florida Maternal, Infant and Early Childhood Home Visiting (MIECHV) Initiative funds perinatal home visiting for pregnant women and families with infants throughout the state. Florida MIECHV has shown resilience to disasters and times of crises in the past, while generating a culture of adaptation and continuous quality improvement among local implementing agencies. Florida MIECHV responded to the COVID-19 pandemic crisis within the first few days of the first reported case in Florida by providing guidance on virtual home visits and working remotely. ASSESSMENT: Findings highlight the role of administrative leadership and communication, staff willingness/morale, logistical considerations, and the needs of enrolled families who face hardships during the pandemic such as job loss, limited supplies, food insecurity, technology limitations, and stress. Home visitors support enrolled families by connecting them with resources, providing public health education and delivering evidence-based home visiting curricula virtually. They also recognized the emotional burden surrounding COVID-19 impacts and uncertainties along with achieving work-life balance by caring for their own children. CONCLUSION: This evaluation helped in understanding the impact of the pandemic on this maternal and child health program and fundamentals of transition to virtual home visiting services. Virtual home visiting appears to be feasible and provides an essential connection to supports for families who may not otherwise have the means or knowledge to access them.

Structure of a bacterial type IV secretion core complex at subnanometre resolution.

Type IV secretion (T4S) systems are able to transport DNAs and/or proteins through the membranes of bacteria. They form large multiprotein complexes consisting of 12 proteins termed VirB1-11 and VirD4. VirB7, 9 and 10 assemble into a 1.07 MegaDalton membrane-spanning core complex (CC), around which all other components assemble. This complex is made of two parts, the O-layer inserted in the outer membrane and the I-layer inserted in the inner membrane. While the structure of the O-layer has been solved by X-ray crystallography, there is no detailed structural information on the I-layer. Using high-resolution cryo-electron microscopy and molecular modelling combined with biochemical approaches, we determined the I-layer structure and located its various components in the electron density. Our results provide new structural insights on the CC, from which the essential features of T4S system mechanisms can be derived.

Structure of the outer membrane complex of a type IV secretion system.

Type IV secretion systems are secretion nanomachines spanning the two membranes of Gram-negative bacteria. Three proteins, VirB7, VirB9 and VirB10, assemble into a 1.05 megadalton (MDa) core spanning the inner and outer membranes. This core consists of 14 copies of each of the proteins and forms two layers, the I and O layers, inserting in the inner and outer membrane, respectively. Here we present the crystal structure of a approximately 0.6 MDa outer-membrane complex containing the entire O layer. This structure is the largest determined for an outer-membrane channel and is unprecedented in being composed of three proteins. Unexpectedly, this structure identifies VirB10 as the outer-membrane channel with a unique hydrophobic double-helical transmembrane region. This structure establishes VirB10 as the only known protein crossing both membranes of Gram-negative bacteria. Comparison of the cryo-electron microscopy (cryo-EM) and crystallographic structures points to conformational changes regulating channel opening and closing.

Type IV secretion machinery: molecular architecture and function.

Bacteria have evolved several secretion machineries to bring about transport of various virulence factors, nutrients, nucleic acids and cell-surface appendages that are essential for their pathogenesis. T4S (Type IV secretion) systems are versatile secretion systems found in various Gram-negative and Gram-positive bacteria and in few archaea. They are large multisubunit translocons secreting a diverse array of substrates varying in size and nature from monomeric proteins to nucleoprotein complexes. T4S systems have evolved from conjugation machineries and are implicated in antibiotic resistance gene transfer and transport of virulence factors in Legionella pneumophila causing Legionnaires' disease, Brucella suis causing brucellosis and Helicobacter pylori causing gastroduodenal diseases. The best-studied are the Agrobacterium tumefaciens VirB/D4 and the Escherichia coli plasmid pKM101 T4S systems. Recent structural advances revealing the cryo-EM (electron microscopy) structure of the core translocation assembly and high-resolution structure of the outer-membrane pore of T4S systems have made paradigm shifts in the understanding of T4S systems. The present paper reviews the advances made in biochemical and structural studies and summarizes our current understanding of the molecular architecture of this mega-assembly.

On the design of a compact emergency mechanical ventilator with negative expiratory exit pressure for COVID-19 patients.

The present work deals with the design of a cylinder-piston arrangement to deliver the required tidal volume (TV) of air to the patient through the respiratory tract especially in the setting of severe acute respiratory syndrome corona virus 2 (SARS CoV-2) or corona virus disease (COVID-19). The design ensures that only the desired volume of air is delivered in each breath and a negative pressure is retained at the delivery point in a separate cylinder. The frequency of piston motion is the same as that of the average human respiratory rate (RR). The effect of negative pressure on time of evacuation under the present condition has been verified. The present design provides a compact ventilator unit with a surface area of 0.8 × 0.4 m2 with a minimal power requirement of 116.48 W. An RR of 16 is obtained with a volume flow rate in lit/s by using a twin cylinder arrangement with bore diameter 0.1 m and length 0.4 m. The ratio of inspiration time to expiration time is designed to be 1:2 by controlling the stroke frequency as 16 and piston speed 0.32 m/s. The present design provides promising quantitative information on the design of an automated continuous mechanical ventilator (CMV), which is different from bag mask valve (BMV) operated ventilators, and on preventing and minimising barotrauma.

An evaluation of factors associated with safe infant sleep practices among perinatal home visiting participants in Florida, United States.

Sleep-related deaths are a leading cause of infant mortality in Florida. The American Academy of Pediatrics recommends placing infants to sleep on their back, alone, and without soft bedding. Compliance with these guidelines varies among parents. This evaluation examined the rates of safe infant sleep practices and associated factors among 1985 participants enrolled in Florida Maternal, Infant, and Early Childhood Home Visiting (FL MIECHV) programs during 2017-2019. Participant- and program-level variables were examined in relation to three sleep practices: infant position, bedding, and bed-sharing at 2-3 months to determine which factors were associated with high rates of safe sleep outcomes. Analyses included univariate descriptive statistics, bivariate statistics, and multivariable logistic regression. Most caregivers (70%) reported always placing their babies to sleep on their back, alone, and without soft bedding. Factors such as primary language, race, education, housing situation, and year the Safe Baby™ curriculum implemented were significantly associated with safe infant sleep practices. Bearing this in mind, FL MIECHV can tailor safe sleep education, messaging, and strategies to support participants at highest risk. Recent adoption of the Safe Baby™ curriculum, and associated staff training, was an important factor influencing parents' infant sleep practices.

Polynucleotide phosphorylase activity may be modulated by metabolites in Escherichia coli.

RNA turnover is an essential element of cellular homeostasis and response to environmental change. Whether the ribonucleases that mediate RNA turnover can respond to cellular metabolic status is an unresolved question. Here we present evidence that the Krebs cycle metabolite citrate affects the activity of Escherichia coli polynucleotide phosphorylase (PNPase) and, conversely, that cellular metabolism is affected widely by PNPase activity. An E. coli strain that requires PNPase for viability has suppressed growth in the presence of increased citrate concentration. Transcriptome analysis reveals a PNPase-mediated response to citrate, and PNPase deletion broadly impacts on the metabolome. In vitro, citrate directly binds and modulates PNPase activity, as predicted by crystallographic data. Binding of metal-chelated citrate in the active site at physiological concentrations appears to inhibit enzyme activity. However, metal-free citrate is bound at a vestigial active site, where it stimulates PNPase activity. Mutagenesis data confirmed a potential role of this vestigial site as an allosteric binding pocket that recognizes metal-free citrate. Collectively, these findings suggest that RNA degradative pathways communicate with central metabolism. This communication appears to be part of a feedback network that may contribute to global regulation of metabolism and cellular energy efficiency.

An Agrobacterium VirB10 mutation conferring a type IV secretion system gating defect.

Agrobacterium VirB7, VirB9, and VirB10 form a "core complex" during biogenesis of the VirB/VirD4 type IV secretion system (T4SS). VirB10 spans the cell envelope and, in response to sensing of ATP energy consumption by the VirB/D4 ATPases, undergoes a conformational change required for DNA transfer across the outer membrane (OM). Here, we tested a model in which VirB10 regulates substrate passage by screening for mutations that allow for unregulated release of the VirE2 secretion substrate to the cell surface independently of target cell contact. One mutation, G272R, conferred VirE2 release and also rendered VirB10 conformationally insensitive to cellular ATP depletion. Strikingly, G272R did not affect substrate transfer to target cells (Tra(+)) but did block pilus production (Pil(-)). The G272R mutant strain displayed enhanced sensitivity to vancomycin and SDS but did not nonspecifically release periplasmic proteins or VirE2 truncated of its secretion signal. G272 is highly conserved among VirB10 homologs, including pKM101 TraF, and in the TraF X-ray structure the corresponding Gly residue is positioned near an α-helical domain termed the antenna projection (AP), which is implicated in formation of the OM pore. A partial AP deletion mutation (ΔAP) also confers a Tra(+) Pil(-) phenotype; however, this mutation did not allow VirE2 surface exposure but instead allowed the release of pilin monomers or short oligomers to the milieu. We propose that (i) G272R disrupts a gating mechanism in the core chamber that regulates substrate passage across the OM and (ii) the G272R and ΔAP mutations block pilus production at distinct steps of the pilus biogenesis pathway.

Two-step and one-step secretion mechanisms in Gram-negative bacteria: contrasting the type IV secretion system and the chaperone-usher pathway of pilus biogenesis.

Gram-negative bacteria have evolved diverse secretion systems/machineries to translocate substrates across the cell envelope. These various machineries fulfil a wide variety of functions but are also essential for pathogenic bacteria to infect human or plant cells. Secretion systems, of which there are seven, utilize one of two secretion mechanisms: (i) the one-step mechanism, whereby substrates are translocated directly from the bacterial cytoplasm to the extracellular medium or into the eukaryotic target cell; (ii) the two-step mechanism, whereby substrates are first translocated across the bacterial inner membrane; once in the periplasm, substrates are targeted to one of the secretion systems that mediate transport across the outer membrane and released outside the bacterial cell. The present review provides an example for each of these two classes of secretion systems and contrasts the various solutions evolved to secrete substrates.

The RNA degradosome: life in the fast lane of adaptive molecular evolution.

In Escherichia coli, the multi-enzyme RNA degradosome contributes to the global, posttranscriptional regulation of gene expression. The degradosome components are recognized through natively unstructured "microdomains" comprising as few as 15-40 amino acids. Consequently, the degradosome might experience a comparatively smaller number of evolutionary constraints, because there is little requirement to maintain a folded state for the interaction sites. New regulatory properties of the degradosome could arise with relative rapidity, because partners that modify its function could be recruited by quickly evolving microdomains. The unusual combination of the centrality of RNA degradation in gene expression and the generality of natively unstructured microdomains in recognition can fortuitously confer a capacity for efficacious adaptive change to degradosome-like assemblies in eubacteria.

An Analysis of Maternal, Social and Household Factors Associated with Childhood Anemia.

Anemia is highly prevalent in all strata of populations in India, with established evidence of intergenerational anemia. The state of Madhya Pradesh was selected to study childhood anemia as the population is mostly rural, with many tribal districts, and has the highest infant mortality rate in India. This study aims to understand the maternal, social and household factors that affect anemia among children aged 6 months to 5 years by analyzing the the National Family Health Survey (NFHS) conducted in 2015-2016. Children aged 6-59 months with estimated hemoglobin levels were included in this study. Bivariate and multivariable analyses were conducted to understand associations between childhood anemia and various socioeconomic factors. Two models to understand the presence of anemia and the levels of anemia were computed. Higher likelihood of having severe childhood anemia was observed among children of younger mothers (15- to 19-year-old mothers (adjusted odds ratio (aOR) 2.08, 95% confidence interval (CI): 1.06, 4.06, less educated (uneducated mothers aOR 2.25, 95% CI 1.13, 4.48) and belonged to a scheduled tribe (aOR 1.88, 95% CI 1.07, 3.29). Strong associations between anemia in mothers and their children suggest intergenerational anemia which has long-term effects. Malnourished children (severe stunting aOR 3.19, 95% CI 2.36, 4.31) and children born with very low birth weight (aOR 4.28, 95% CI 2.67, 6.87) were more likely to have anemia. These findings strongly suggest more proactive interventions including prenatal healthcare for women and monitoring of the nutrition children at the community level to combat childhood anemia. Evaluations of existing programs should be conducted to understand the gaps in reducing anemia and malnutrition in children.

Protein oligomerization in the bacterial outer membrane (Review).

The formation of homo-oligomeric assemblies is a well-established characteristic of many soluble proteins and enzymes. Oligomerization has been shown to increase protein stability, allow allosteric cooperativity, shape reaction compartments and provide multivalent interaction sites in soluble proteins. In comparison, our understanding of the prevalence and reasons behind protein oligomerization in membrane proteins is relatively sparse. Recent progress in structural biology of bacterial outer membrane proteins has suggested that oligomerization may be as common and versatile as in soluble proteins. Here we review the current understanding of oligomerization in the bacterial outer membrane from a structural and functional point of view.

Recognition and cooperation between the ATP-dependent RNA helicase RhlB and ribonuclease RNase E.

The Escherichia coli protein RhlB is an ATP-dependent motor that unfolds structured RNA for destruction by partner ribonucleases. In E. coli, and probably many other related gamma-proteobacteria, RhlB associates with the essential endoribonuclease RNase E as part of the multi-enzyme RNA degradosome assembly. The interaction with RNase E boosts RhlB's ATPase activity by an order of magnitude. Here, we examine the origins and implications of this effect. The location of the interaction sites on both RNase E and RhlB are refined and analysed using limited protease digestion, domain cross-linking and homology modelling. These data indicate that RhlB's carboxy-terminal RecA-like domain engages a segment of RNase E that is no greater than 64 residues. The interaction between RhlB and RNase E has two important consequences: first, the interaction itself stimulates the unwinding and ATPase activities of RhlB; second, RhlB gains proximity to two RNA-binding sites on RNase E, with which it cooperates to unwind RNA. Our homology model identifies a pattern of residues in RhlB that may be key for recognition of RNase E and which may communicate the activating effects. Our data also suggest that the association with RNase E may partially repress the RNA-binding activity of RhlB. This repression may in fact permit the interplay of the helicase and adjacent RNA binding segments as part of a process that steers substrates to either processing or destruction, depending on context, within the RNA degradosome assembly.

Recognition of enolase in the Escherichia coli RNA degradosome.

In Escherichia coli, the glycolytic enzyme enolase is a component of the RNA degradosome, which is an RNase E mediated assembly involved in RNA processing and transcript turnover. The recruitment of enolase by the RNA degradosome has been implicated in the turnover of certain transcripts, and it is mediated by a small segment of roughly a dozen residues that lie within a natively unstructured sub-domain of RNase E. Here, we present the crystal structure of enolase in complex with its recognition site from RNase E at 1.6A resolution. A single molecule of the RNase E peptide binds asymmetrically in a conserved cleft at the interface of the enolase dimer. The recognition site is well conserved in RNase E homologues in a subfamily of the gamma-proteobacteria, including enzymes from pathogens such as Yersinia pestis, Vibrio cholera and Salmonella sp. We suggest that enolase is recruited into putative RNA degradosome machinery in these bacilli, where it plays common regulatory functions.

Structure of the regulatory apparatus of a calcium-dependent protein kinase (CDPK): a novel mode of calmodulin-target recognition.

Calcium-dependent protein kinases (CDPKs) are a class of calcium-binding sensory proteins that are found in plants and certain protozoa, including the causative agent of malaria, Plasmodium falciparum. CDPKs have diverse regulatory functions, including involvement in the triggering of the lytic cycle of malarial infection. CDPKs contain an autoinhibitory junction (J) region whose calcium-dependent interaction with the tethered regulatory calmodulin-like domain (CaM-LD) activates the catalytic kinase domain. We report here the X-ray crystal structure of the J-CaM-LD region of CDPK from Arabidopsis thaliana (AtCPK1), determined to 2.0 A resolution using multiple-wavelength anomalous dispersion (MAD). The structure reveals a symmetric dimer of calcium-bound J-CaM-LD with domain-swap interactions, in which the J region of one protomer interacts extensively with the carboxy-terminal EF-hand domain (C-lobe) of the partner protomer. However, as the J-CaM-LD is monomeric in solution, the activated monomer was modelled to account for the intra-molecular recognition of the two domains. While the J-CaM-LD segment mimics certain aspects of target motif recognition by CaM other features are specific to CDPKs, in particular the combination of the strong interaction between the N and C-lobes of the CaM-LD and the exclusive use of only the C-lobe in the recognition of the covalently tethered target region. Combined with our previous observations showing that there is likely to be strong interactions between this tethered J region and the CaM-LD even at basal Ca(2+) concentrations, the new structural data indicate that the response to calcium of CDPKs is clearly unique among the CaM family.

Studies of the RNA degradosome-organizing domain of the Escherichia coli ribonuclease RNase E.

The hydrolytic endoribonuclease RNase E, which is widely distributed in bacteria and plants, plays key roles in mRNA degradation and RNA processing in Escherichia coli. The enzymatic activity of RNase E is contained within the conserved amino-terminal half of the 118 kDa protein, and the carboxy-terminal half organizes the RNA degradosome, a multi-enzyme complex that degrades mRNA co-operatively and processes ribosomal and other RNA. The study described herein demonstrates that the carboxy-terminal domain of RNase E has little structure under native conditions and is unlikely to be extensively folded within the degradosome. However, three isolated segments of 10-40 residues, and a larger fourth segment of 80 residues, are predicted to be regions of increased structural propensity. The larger of these segments appears to be a protein-RNA interaction site while the other segments possibly correspond to sites of self-recognition and interaction with the other degradosome proteins. The carboxy-terminal domain of RNase E may thus act as a flexible tether of the degradosome components. The implications of these and other observations for the organization of the RNA degradosome are discussed.

Management of a nonvital young permanent tooth by pulp revascularization.

This report presents the case of a 10-year-old patient with a nonvital young permanent tooth which was managed by pulp revascularization. Following disinfection of the canal by irrigation with NaOCl and use of a triantibiotic paste, a scaffold was created by inducing the formation of a blood clot within the canal. At the subsequent follow-up visits, the patient was asymptomatic, with normal response to percussion, normal periodontal probing depths, and no abnormal mobility. The radiographs showed evidence of continued apical root development with increase in root length, signs of apical closure and increase in thickness of dentinal walls. Thus, this case adds to the growing evidence supporting the revascularization approach as an option for management of nonvital young permanent teeth. How to cite this article: Chandran V, Chacko V, Sivadas G. Management of a Nonvital Young Permanent Tooth by Pulp Revascularization. Int J Clin Pediatr Dent 2014;7(3):213-216.