The growth of expertise in molecular techniques, their application to clinical evaluations, and the establishment of databases with molecular genetic information has led to greater insights into the roles of molecular processes related to gene expression in neurodevelopment and functioning. The goal of this review is to examine new insights into messenger RNA transcription, translation, and cellular protein synthesis and the relevance of genetically determined alterations in these processes in neurodevelopmental, cognitive, and behavioral disorders. Precise stimuli for gene expression in neurons remain to be elucidated. Early studies revealed that neurotransmitter activation of receptors triggered intracellular signaling. Abundant evidence of the importance of epigenetic factors and chromatin modifiers in gene expression in brain development and function can be derived from data on the high frequency of mutations in chromatin modifiers in neurodevelopmental, neurocognitive, and neurobehavioral disorders , 2.The transcription start site was noted to be embedded in the core promoter, and the core promoter was defined as including sequence approximately 50 bp upstream and 50 bp downstream of the transcription start site.Sequence elements in the promoter region act as binding sites for transcription factors, including general transcription factors and transcription cofactors, sequence elements for enhancer binding and the promoter region forms an anchoring site for RNA polymerase II.Specific sequence elements occur in some, but not all, promoters; these included the TATA box and a sequence element defined as the initiator motif. Specific features of promoter-associated chromatin have been defined that are strongly associated with transcription; these include H3K4me3 and H3K27ac. Transcription initiation at core promoters is defined as a stepwise process that includes recruitment to the promoter of general transcription factors TFIID, TFIIA, TFIIB, and RNA polymerase II, followed by recruitment of general transcription factors TFIIE and TFII H.Specific histone methyl transferases and histone demethylase interact with enhancers. Enhancer activation was considered to be a stepwise process, and active enhancers could be transcribed by RNA polymerase II to yield enhancer RNA (eRNA) transcripts. The eRNA level indicated the degree of activation of the enhancer. The eRNA then interacted with the gene promoter, and this interaction was mediated by cohesin and mediator complexes. Highly active enhancers were sometimes referred to as super-enhancers, and these were often several kilobases in length.In It is important to note that enhancers may also function in trans relative to the genes they regulate. Enhancers that function in cis relative to the genes they regulate may employ chromatin looping in gene interactions.There is now evidence that a large proportion of the human genome is transcribed, and increasingly, long non-coding RNAs are identified. Linc RNAs were reported to be primarily present in the nucleus, and their functions were reported to be regulating chromatin topology and formation of scaffolds for binding of proteins. Linc RNAs were reported to stabilize chromatin and to also play roles in chromatin looping.Specific architectural proteins including CTCF CCCTC-binding factor, YY1 (transcription factor), and cohesin complex subunits were reported to be anchors of chromatin loops.1.Chromatin loops were shown to be important features of the vertebrate genome and to serve to bring enhancers into contact with promoters. The importance of this process was demonstrated in erythroid cells, and a specific upstream locus control region was brought into contact with the beta globin gene locus . ImportaCore elements of the cohesin complex are presented in Mutations in SMC1, SMC3, RAD21, and NIPBL have been reported to lead to Cornelia de Lange syndrome associated with developmental defects in a number of systems .Topologically associated domains (TADS) were defined as sub-megabase-sized domains in which preferential intra-domain interactions occurred, indicating that DNA elements within the TADS interact . GenomicSpecific architectural proteins including CTCF CCCTC-binding factor, YY1 (transcription factor), and cohesin complex subunits were reported to be anchors of chromatin loops.Developmental defects can also result from structural chromosome changes that disrupt TADS.There is evidence that chromatin loop formation organized by cohesin and CCCTC binding factor (CTCF) plays important roles in the regulation of gene transcription and in the organization of the 3-dimensional genome. Chromosomes, chromatin, chromatin regulators, and architectural proteins were reported to be involved in the formation of the 3-dimensional genome that has been shown to play an important role in gene expression.Other factors that promote the cohesion of sister chromatids include ESCO 1, ESCO2 establishment of sister chromatid cohesion N-acetyltransferase 1 and 2, PDS5 cohesin associated factor B, and Sororin also known as CDCA5 cell cycle division-associated 5.The length of the 3′UTR region upstream of the polyadenylation site utilized influences availability of sites for binding of microRNAs and for binding of other RNA-binding proteins.Factors in the polyadenylation complex were also noted to be more abundant in proliferating stem cells and in pluripotent stem cells.Use of proximal polyadenylation sites was also noted when neuronal cells were activated by membrane depolarization. Activation of mammalian target of rapamycin (mTOR) signaling was also reported to lead to the generation of transcripts with shorter 3′-UTRs.Core splicing elements were noted to include 5′ and 3′ splice sites at exon intron junctions, branch points, and polypyrimidine tracts within introns. In addition, splicing is influenced by enhancers and silencers, nucleotide elements that can have trans effects in splicing machinery. Splicing is also dependent on appropriate function of components of the spliceosome.Genome-wide association studies in autism demonstrated an autism spectrum disorder-associated variant in PTBP2 polypyrimidine tract binding protein 2 that binds to intronic polypyrimidine clusters in pre-mRNA molecules and is implicated in controlling the assembly of other splicing-regulatory proteins.The composition of the highly dynamic spliceosome was reviewed by The spliceosome is described as a multi-megadalton ribonucleoprotein complex. It contains small nuclear ribonucleoproteins (snRNPs) and many other proteins. Core elements recognized by the spliceosome include the 5′ donor site GU dinucleotides, the branch point followed by the polypyrimidine tract and subsequently the AG dinucleotide at 3′ splice acceptor site.Alternative splicing was reported to be highly regulated and to be responsive to developmental and environmental stimuli . AlternaSNRPN gene located in chromosome 15q11.2 in the region involved in Prader–Willi syndrome is reported to encode a component of the snRNP complex, expressed from the paternally derived chromosome. It functions in pre-mRNA processing and may contribute to tissue-specific alternative splicing.The Numerous studies in humans with autism have revealed altered neuronal excitation–inhibition ratios resulting from altered neuronal activity and synaptic function. Many different gene defects have been reported to be associated with autism manifestations. In addition, microarray analyses and RNA sequence analyses have revealed altered patterns of gene expression. Misregulation and reduced transcription of the RBFOX family of RNA-binding splice regulators were reported by The specific nSR100 defects investigated by The activity of nSR100 was shown to be important in the inclusion of microexons in the transcripts of EIF4G component of the transcription initiation complex, in MEF2C transcription factor transcripts, and in transcripts shown to be aberrant in autism including ANK2, NBEA (neurobeachin), NRXN2 (neurexin 2), and in SHANK 1 (SH3 and multiple ankyrin repeat domains 1).Importantly, they postulated that nSR100.SRRM4 could potentially be a target for diagnostics and therapeutic intervention in autism.Early-switch exons were reported to be enriched in the transcripts of genes that encoded ion channels and in genes that encoded products related to transmembrane transport and synaptic transmission. Late-switch exons were enriched in transcripts of genes involved in cytoskeletal remodeling and in the formation of synaptic connections.In mouse brain, Posttranscriptional mechanisms, including alternative splicing, in neural developments, maturation, and plasticity, were reviewed by Information on the functional roles of alternative splicing in particular genes has sometimes been derived from information on pathology and disease manifestation that result from splicing mutations in specific genes.Insights into splice regulation have also been obtained through studies on specific nucleotide elements that interact with RNA-binding proteins and the precise positions of these binding elements relative to alternate exons.They noted that the presence of cell type heterogeneity in tissues complicates alternative splicing studies. In recent years, transcription studies are increasingly carried out on single cell or on samples with a uniform cell population.Another important factor to consider is that specific RNA-binding proteins require modifications, and changes in these modifications can impact their binding properties.Drosophila and studies of the transmembrane receptor Dscam1. Evidence was obtained indicating that extensive alternative splicing of Dscam 1 occurred, leading to the generation of products with different numbers of Ig domain inclusions. There was evidence that in dendrite arbor formation, neurites expressing the same number of repeat containing Dscam1 molecules formed connections with each other while dendrites each expressing different Dscam1 isoforms that differed in the number of Ig repeat domain repulse each other and failed to connect. The Dscam1 gene in Drosophila is the paralog of a human gene DSCAM that is impacted in Down syndrome.They considered whether transcript splicing patterns impacted circuit wiring in the brain and noted that there was evidence that alternate splicing of specific transcripts impacts axonal guidance and synapse formation. This information was derived primarily from studies in The DCC guidance receptor, also known as DCC netrin receptor, was shown to undergo alternate splicing events in mice, and the specific isoform expressed impacted axon guidance and interaction.Transcripts of neurexin, the synaptic adhesion molecule, were reported to exhibit significant differences based on splice site usage differences.Transcripts of KCNMA1, the potassium calcium-activated channel, were also reported to exhibit splicing differences, leading to protein products that differentially influence neuronal excitability.Mutations in genes that encode sodium ion channels are important causes of epilepsy. Mutations in the SCNA genes are reported to be associated with Dravet syndrome, an autosomal dominant disorder associated with encephalopathy, epilepsy, and developmental delay.Through studies in autism and developmental disabilities, they provided evidence that putative loss of function variants in weakly expressed exons in neuronal cell types has the same level of significance as synonymous variants.They noted that no information tools currently exist for incorporation of transcript analysis into variant interpretation.MRNA degradation processes include mechanisms involved in degradation of normal MRNA and also additional processes involved in degradation of mutant MRNA. Degradation processes include endonucleolytic cleavage, exonucleolytic cleavage, de-adenylation, and exosome activity. Particular processes are involved in the degradation of mRNA forms with premature termination codons.Exosomes are involved in trimming and degradation of mRNAs. The presence of AU-rich elements in the 3′-UTR of mRNA facilitates binding to exosomes. Importantly, mutations in specific exosome component EXOSC3 were reported to be associated with the neurodevelopmental disorder pontocerebellar hypoplasia. Mutations in the exosome component EXOSC8 was associated with a disorder in children in which cerebellar hypoplasia, hypomyelination, and spinal muscular atrophy occurred.There is evidence that exosome activity occurs in the nucleus and in the cytoplasm. MRNA to be degraded is reported to be guided to the exosome in the nucleus by a complex referred to as the TRAMP complex. In the cytoplasm, the SKI complex guides MRNA for degradation to the exosome. The MTR4 (MTREX) helicase complex was reported to be important for mRNA degradation in humans.Long non-coding RNAs were postulated by MRNA passes from nucleus to cytoplasm through nuclear pore complexes. This complex is composed of multiple proteins including nucleoporins, and energy for transport through nuclear pores is dependent on guanine triphosphate. Exportin proteins also facilitate transport out of the nucleus. In recent years, there are reports of nucleoporin defects in specific neurodevelopmental disorders. RANBP2 is a large protein that localizes to the nuclear pore complex. Defects in RANBP2 have been reported to predispose to familial encephalopathy . FichtmaThe preinitiation complex refers to a complex that includes guanosine triphosphate (GTP) bound to eIF2 and methionyl initiator tRNA, eIF3, eIF5, eIF1, eIF1A 40S ribosome subunit. The PIC complex was reported to scan mRNA until the AUG initiation codon was encountered. Subsequently, hydrolysis of the eIF2-bound GTP occurs, leading to the release of many of the bound initiation factors. This facilitates recruitment of GTP-bound eIF5B and the 80S binding, and the remaining bound eIF factors are released. The aminoacyl-tRNA-binding site on ribosomes is exposed, allowing translation extension to occur.Another important factor involved in controlling the degree of cellular protein synthesis is the integrated stress response .Specific translation initiation complex subunits that recognize the 3-end of the mRNA include eif4G and eif4E. Additional initiation factors that serve to bring the mRNA to the ribosome include eiF4E, eiF4G, eiF4A, and eif4b, and eiF4G was reported to also interact with eiF3.Following this binding, the 40S ribosome subunit with bound eif5 and initiator methionyl tRNA scans the mRNA for the AUG initiator codon. Hydrolysis of GTP on eif2 occurs through the activity of eiF5; the eiF2 is then released, and the 60s subunit of the ribosome joins the 40s ribosomal subunit and translation can then begin.In 2012, Microsatellite nucleotide repeat expansions were reported to be associated with more than 49 different diseases that impact the central and peripheral nervous systems. There is growing evidence that the repeat sequence regions can be transcribed and translated, giving rise to aberrant peptides and proteins. The first RAN translation process was recognized in spin cerebellar ataxia cases associated with a CAG repeat expansion. There is some evidence that aberrant dipeptides and short proteins derived from aberrant translation can accumulate in inclusions.Translation involves aminoacyl tRNAs being escorted to ribosomes by the GTP-bound elongation factors, e.g., eEf1, eEf1a. On recognition of the cognate nucleotide codon in the mRNA, the specific aminoacyl-tRNA interacts with the ribosome and polypeptide, and the GTP on the elongation factor is hydrolyzed. The elongation process involves movement of the ribosomes along three nucleotides of the mRNA with each addition of an amino acid. Elongation proceeds until a termination codon is encountered in the mRNA transcript.Each aminoacyl-tRNA synthetase couples a specific amino acid. MRI abnormalities, psychomotor retardation, microcephaly, hypotonia, seizures, and encephalopathy were frequently encountered. Failure to thrive was a commonly encountered early manifestation in children. Other manifestations that were encountered in some patients included liver, bone marrow, and gastrointestinal defects.They defined tRNAs as adaptors in the translation process. Multistep maturation processes are required for tRNAs to be functional. In addition, there is evidence that TRNAs have other functions.In reviewing the tRNA life cycle, In the mitochondria, 22 tRNA genes were reported to be active. Products for modifying mitochondrial tRNAs were reported to be encoded in the nucleus and to require transport into mitochondria. Following modification, TRNAs occur in the cytoplasm, mitochondria, and endoplasmic reticulum. Specific transporters of TRNAs included exportin 7 encoded by the XPO7 gene.Twenty different aminoacyl tRNA synthetases occur, each recognizing a specific amino acid.Childhood neurological disorders associated with impaired biogenesis of nuclear TRNAs are noted to lead to hypomyelinating leukodystrophies and pontocerebellar hypoplasia. Leukodystrophies have also been reported to result from POLR3A or POLR3B defects. POLR3A and POLR3B encode the component of RNA polymerase III that is involved in the transcription of tRNA genes.Pontocerebellar hypoplasia can result from defects in functions of the products of the TSEN genes including TSEN2, TSEN15, TSEN34, and TSEN54 that encode tRNA splicing endonucleases.Neurological disorders can also result from defective functions of enzymes that charge tRNAs, namely, TRNA synthetases. Specific syndromic disorders due to defects in aminoacyl-tRNA synthetases include forms of Charcot–Marie–Tooth disease that is characterized by impaired peripheral nerve functions primarily in axons that often first present with abnormalities in the legs. Defects in any one of more than 70 different genes have been reported to lead to this disorder. The PMP2 gene that encodes peripheral myelin protein 22 is the most commonly involved. Defects leading to Charcot–Marie–Tooth disease have been identified in six different aminoacyl-tRNA synthetases, alanyl-t-RNA synthetase 1 (AARS1), methionyl-tRNA synthetase 1 (MARS1), histidyl-tRNA synthetase 1 (HARS1), tyrosyl-tRNA synthetase 1 (YARS), lysyl-tRNA synthetase 1 (KARS), and glycyl-tRNA synthetase 1 (GARS).This combined phenotype can also arise due to a defective function of mitochondrial methionyl-tRNA transformylase (MTFmt) that is required for initiation of translation in the mitochondria.Microcephaly with cerebellar atrophy was reported to result from defects in glutaminyl-tRNA synthetase 1 (QARS1) or defects in seryl-tRNA synthetase 1 (SARS1).Premature termination codons in mRNA elicit a mechanism referred to as nonsense-mediated decay that involves a surveillance complex composed of RENT1 also referred to as UPF1 RNA helicase and ATPase and peptide release factor also known as ERF3A.The proteins eEF1A1 and eEF1A2 were noted to constitute central components of the complex with eEF1A2 occurring primarily in neurons and eEF1A1 widely present. The EEF1A gene was reported to be mutated in specific cases of autism. The eEF1A complex was reported to function to deliver aminoacyl-associated tRNAs to the ribosome to generate the polypeptide chain. Energy for this process is provided by GTP.Deletion of an exon in the EEF1B2 gene was reported to be present in three siblings with intellectual disability in one family. EEF1D mutations have been reported to be associated with intellectual disability in members of one family. Different VARS gene mutations were reported to occur in two different families with intellectual disability.They noted that advances in molecular techniques have expanded insights into factors involved in translation and its termination. One of these techniques includes ribosome profiling. This technique is designed to determine quantitative measures of protein synthesis. A specific aminoacyl-tRNA synthetase must select the correct amino acid, and it must select the correct tRNA from the TRNA pool. Specific editing processes take place. In pretransfer editing, an incorrectly bound amino acid can be removed from the aminoacyl-tRNA synthetase by hydrolysis. Mischarged tRNAs can also be edited. These pre- and posttranslation editing processes require the existence of proofreading mechanisms .Translation Elongation factor mutations in EEF1A2 were reported in cases of epilepsy, ataxia, intellectual disability, and autistic behavior by Translation termination factors were designated eRF, and genes were designated ERF or ETF1. Termination proteins included eRF1, eTF1 (eRF3); a gene encoding etf3 was not found in the human gene database. It is important to note that nomenclature for translation factors remains complex.A specific process referred to as ribosome quality control (RQC) is initiated when a ribosome stalls during the process of translation. When ribosomes stall, the aberrant or shortened polypeptide chain undergoes proteosomal degradation. in vitro studies to study the effects of regulatory regions and target genes.Their study focused on analyses of haplotypes of defined regulatory regions that were located in cis to the target gene. They also utilized The study was based on the hypothesis that nucleotide variants, the haplotype, in regulatory regions impact penetrance; specific haplotypes reduced penetrance, while other haplotypes were disadvantageous and increased penetrance, while other haplotypes were advantageous and individuals with the advantageous haplotype showed few manifestations of the disease despite the presence of deleterious mutations in the target gene. They also postulated that the deleterious haplotypes were rare in the general population. Patients with manifestations of the disease caused by deleterious mutations in a specific disease would have an overrepresentation of specific regulatory haplotypes relative to the frequency of those haplotypes in the general population.Among the results of their study was evidence for a regulatory region haplotype that modified the phenotype of Birt–Hogg–Dubé syndrome. This syndrome was reported by It is however important to note that there are many other factors that can influence the clinical expressivity and penetrance of a specific deleterious variant. In a review in 2013, Posttranslational modifications are often important in regulating protein functions and in determining the locations of specific proteins in specific subcellular organelles. In addition, specific factors influence the correct three-dimensional folding of proteins that may be required for their function.SCN1B played roles in epilepsy. Sodium ion channel proteins occur in neuronal soma and in dendrites and to have high density in the axon initial segment and in the myelinated node of Ranvier. Sodium ion channels are reported to initiate electrical neuronal action potentials.Five different genes that encode alpha subunits of sodium ion channels have been reported to have mutations that lead to epilepsy; these include SCN1A, SCN2A, SCN3A, SCN8A, and SCN9A, and epilepsy-inducing mutations have also been reported in SCN1B.In addition, posttranslational modifications are required for the sodium ion channel functions. The extent to which specific amino acid residues are involved in posttranslational modifications needs to be considered in analyses of the impact of specific mutations on function.The subunit interactions are also impacted by the degree to which posttranslational modifications impact the subunit interactions and protein functions.MS reviewed literature and generated the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Hsp70 chaperones interact with substrate proteins in a coordinated fashion that is regulated by nucleotides and enhanced by assisting cochaperones. There are numerous homologues and isoforms of Hsp70 that participate in a wide variety of cellular functions. This diversity can facilitate adaption or specialization based on particular biological activity and location within the cell. In this review, we highlight two specialized binding partner proteins, Tim44 and IRE1, that interact with Hsp70 at the membrane in order to serve their respective roles in protein translocation and unfolded protein response signalling. Recent mechanistic data suggest analogy in the way the two Hsp70 homologues (BiP and mtHsp70) can bind and release from IRE1 and Tim44 upon substrate engagement. These shared mechanistic features may underlie how Hsp70 interacts with specialized binding partners and may extend our understanding of the mechanistic repertoire that Hsp70 chaperones possess. They are involved in a wide variety of cellular activities that includes folding of newly synthesized proteins; translocation of nascent polypeptides into organelles such as mitochondria, endoplasmic reticulum (ER) and chloroplasts; disassembly of protein complexes and activation of various proteins. Furthermore, in both stressful and non-stressful conditions, they prevent the aggregation of misfolded proteins and coordinate their refolding enabling them to revert to their native state –3. BecauSeveral decades of research have carefully pieced together the mechanism of how Hsp70 chaperones interact with their substrates, a process that is tightly regulated by nucleotides and enhanced by helper proteins known as cochaperones. There are a considerable number of Hsp70 homologues and isoforms present in nature and an even greater variety of assisting cochaperones. This complexity can be explained by the range of biological roles that Hsp70 chaperones are implicated in. Also, this diversity facilitates a certain level of adaption based on cellular location and particular cellular function, and is often referred to as specialization ,4.The role of Hsp70 in driving protein translocation across the inner mitochondrial membrane is a well-described example of how Hsp70 can specialize to fulfil an essential cellular activity. The ER equivalent homologue, known as BiP (binding immunoglobulin protein), can also operate at the membrane to facilitate protein translocation into the ER lumen. Moreover, BiP has been suggested to play a role in the unfolded protein response (UPR), an essential homeostasis pathway that detects and responds to an increase of misfolded protein within the ER –7. How i2.α and SBDβ . The SBDβ consists of an eight-stranded β-sandwich fold which contains the hydrophobic pocket that constitutes the polypeptide-binding site. The SBDα is a helical structure that acts as a lid which can close over the polypeptide-binding pocket [Hsp70 chaperones undergo coordinated movements that facilitate misfolded substrate binding and release. The domain architecture of Hsp70 is generally well conserved across different species and homologues. It comprises a nucleotide-binding domain (NBD) and a substrate-binding domain (SBD) that is connected via a short linker. The NBD is made up of four subdomains that are organized into two lobes which results in the formation of a deep cleft ,9 figur. The actand SBDβ b,c. The g pocket ,15.FiguKon and Koff (low affinity) rate enabling the loading and release of the substrate. Upon ATP hydrolysis and transition to the ADP bound state, Hsp70 undergoes structural re-arrangements that include the positioning of the SBDα lid over the SBD-binding pocket, with the effect of trapping the substrate within the SBD commensurate with a much reduced Kon and Koff rate (high affinity) [d). There are two groups of Hsp70 cochaperones that facilitate the transition between states. (i) J-domain containing proteins that increases hydrolysis thereby stabilizing the substrate interaction. (ii) Nucleotide exchange factors (NEF) that mediate the exchange of ADP to ATP enabling the release of the bound substrate [d). Once maximal ATP hydrolysis is achieved, the effective affinity for the substrate increases by several orders of magnitude in a non-equilibrium fashion and is referred to as ultra-affinity [The binding and release of the substrate is allosterically coupled to nucleotide association and ATP hydrolysis. In the ATP bound state, Hsp70 interacts with substrate protein with a high ffinity) ,12,16–19ubstrate  , NEF Mge1 and the membrane-spanning J-protein Pam18 . mtHsp70The other two essential components of PAM are Tim16 and Tim44, they both act as adaptor proteins that have multiple contacts with the other PAM components. Tim16 contains a degenerate J-like domain which is unable to stimulate Hsp70 activity. It engages Pam18, an association that is important for Pam18 to interact with the conducting channel protein, Tim23 ,29.Tim44 serves as a central hub protein that crucially links the conducting channel to PAM motor proteins on the matrix side of the pore. Tim44 is divided into two domains of approximately equal size and referred to as the N- and C-terminal domains ,31. The During translocation of the pre-sequence through the TIM23 pore, the emerging polypeptide engages mtHsp70. This association releases mtHsp70 from Tim44 and in the process helps to drive through the nascent chain into the matrix. When mtHsp70 is attached to Tim44, it has limited movement due to the constraints of being close to the membrane. The binding of the partially folded pre-sequence polypeptide frees mtHsp70 from its location-based constraints, increasing its entropy, which provides the driving force for translocation  figure .5.The UPR is a cell signalling system that detects the presence of misfolded proteins within the ER and initiates a transcriptional and translational response that aims to restore ER homeostasis. IRE1 is a key primary activator/sensor of the UPR that is conserved from yeast to humans. It spans the ER membrane presenting both luminal and cytosolic domains –7. The lBiP has been suggested to play a role in UPR signalling, although precisely what that may be is subject to debate and contingent upon the various models that have been proposed. A BiP independent UPR activation model suggests that IRE1 directly contacts misfolded proteins, with BiP playing a peripheral role in sequestering inactive IRE1 ,41. By c6.The aforementioned examples of Tim44 and IRE1 are two instances where Hsp70 associates with proteins that are neither a cochaperone (i.e. they do not contain a J-protein), or have NEF ability, or are a misfolded substrate protein. Such intriguing interactions arise due to specialization imposed by the cellular location and the requirement to fulfil a specific biological role. Interestingly, recent data suggests analogy between the well-defined actions of mtHsp70 when binding to Tim44 with that of BiP association to IRE1 in the ‘allosteric' UPR model. These mechanistic similarities are discussed below.7 [First, both BiP and mtHsp70 chaperones are adapted to operate at the membrane due to their ability to bind membrane-spanning or membrane-associated proteins. This enables them to operate in protein translocation and UPR signal induction, as both of these processes occur at the membrane ,6,7,26. 7 . This diSecond, the interaction with Tim44 and IRE1 is primarily mediated by the NBD of mtHsp70 and BiP, respectively , althougThere are several lines of evidence to suggest that IRE1 interacts primarily via BiP NBD. This includes biophysical experiments that measure a direct interaction between BiP NBD and IRE1 LD using microscale thermophoresis . This isin vitro. In cells, there are numerous partially folded proteins that can act as substrate and the addition of ATP was suggested to prime the engagement of misfolded protein leading to dissociation of mtHsp70 from Tim44 [in vitro FRET assay. In these experiments, IRE1 LD and BiP were N-terminally tagged with cyan and yellow fluorescence proteins, which on mixing produced a FRET signal. The subsequent addition of misfolded proteins, but not nucleotides, caused the loss of FRET signal, indicating again the misfolded substrate-dependent dissociation of BiP from IRE1 [Third, the interaction between Hsp70 and specialized binding partners, Tim44 and UPR proteins, are not destabilized by nucleotides. Fourth, that dissociation between specialized binding partners and Hsp70 is dependent on misfolded protein substrate binding and this process is primed by ATP . Both ofom Tim44 ,54. Simiom Tim44 ,56. Howeom Tim44 . Signifiom Tim44 . This warom IRE1 . Moreoverom IRE1 . This inrom IRE1 , althougrom IRE1 .in vitro FRET experiments, measuring the influence of nucleotides on substrate-dependent dissociation of this complex, indicated a clear priming effect in the presence of ATP, but not ADP [Additional  not ADP . The rel not ADP . This ofFor BiP, the binding of IRE1 prevents the association of its cochaperones, ERdj3 and Sil1. ERdj3 and Sil1 are the canonical cochaperones for BiP when performing general protein-folding activities within the ER. This in effect switches BiP from acting as a chaperone to being fully primed to detect misfolded protein. Once BiP binds substrate, it detaches from IRE1 enabling ERdj3 (or Sil1) to interact with BiP to mediate its chaperone substrate cycle . This su7.In this review, we highlight two Hsp70 homologues that have specialized roles in protein translocation and stress signalling at the membrane. In their orthodox activities, which include protein folding and prevention of aggregation, their interaction with substrate protein is well characterized and involves the coordinated movement of their two domains upon ATP binding and hydrolysis, a process that is catalysed by cochaperones. The iterative cycling between such conformations leads to a non-equilibrium high-affinity state that mediates the remodelling of the substrate to help form its native structure. In their alternative specialized roles, they can interact with proteins that are neither cochaperones nor substrates. This type of interaction suggests additional mechanistic features beyond that which is required for interaction with misfolded substrates. In this capacity, we highlight some common features of two specialized binding partner proteins, Tim44 and IRE1, when they interact with their complimentary Hsp70 chaperones, mtHsp70 and BiP. Such features include binding that is mediated primarily via the NBD. Nucleotides do not affect this association but exert their influence by priming the SBD to engage substrate protein. A key defining feature seems to be that dissociation of specialized partner from Hsp70 is dependent on misfolded substrate binding to SBD. The consequences of substrate-dependent dissociation are different for the two homologues as they serve different biological roles. Tim44 presents a platform for mtHsp70 that enables it to bind the nascent translocating polypeptide chain. Subsequent release from the confines of the membrane increases the entropy of the system and provides the driving force for polypeptide insertion into the matrix. For BiP, misfolded-dependent dissociation would presumably trigger oligomerization leading to downstream activation and propagation of the UPR signalling cascade. It would be interesting to observe whether these common mechanistic features can be extended to other specialized binding partners that are positioned away from the membrane. These features may expand our understanding of the mechanistic repertoire that Hsp70 chaperones possess. Thus, in summary, we suggest common mechanistic features for binding and release of BiP and mtHsp70 with specialized binding partners at the membrane.


Few studies have evaluated acitretin as a primary treatment for cutaneous squamous cell carcinoma (CSCC). We report, for the first time, three cases of CSCC successfully treated with acitretin and clarithromycin. A literature review on this subject was also was performed. This case report included three patients with CSCC treated with acitretin and clarithromycin at the First Affiliated Hospital of Fujian Medical University (2008–2019). Patient 1 , patient 2  and patient 3  received 8, 6, and 30 courses of combination therapy. All patients tolerated the adverse effects (pseudotumor cerebri and mucocutaneous dryness) and achieved complete regression within 6 months. Patients 1, 2, and 3 have not experienced recurrence during a 10-, 3-, and 6-year follow-up. Acitretin has limited efficacy as a monotherapy for CSCC. Our experience indicates that combination therapy with acitretin and clarithromycin may be an effective and well-tolerated treatment for unresectable CSCC. Before treatment, the patients were fully informed about the potential advantages and disadvantages of combination therapy with acitretin and clarithromycin, including the possible side effects of these drugs. All patients provided written informed consent for treatment and agreed to attend follow-up visits every 2 weeks. All patients provided written informed consent for data analysis and publication.The present case report represents a retrospective analysis of the medical records of patients with CSCC who were treated with a combination of acitretin and clarithromycin at the Dermatology Department of the First Affiliated Hospital of Fujian between 2008 and 2018. Inclusion criteria consisted of a diagnosis of SCC by pathological biopsy and a history as well as tumor diameter ≥1 cm .A total of 3 patients were involved in our study. Acitretin and clarithromycin were administered together to the patients from the time when skin biopsy confirmed the diagnosis of invasive SCC. We defined oral 30 mg/day of acitretin for 4 weeks and oral 500 mg/day of clarithromycin for 3 weeks as 1-month treatment course. There was no interval time between courses.Once biochemical investigations, including measurements of serum creatinine, cholesterol, triglycerides, and potassium appeared abnormal, the treatment was stopped or the dosage was reduced.2 laser resection of the CSCC was performed after a total of 6 courses of combination therapy, and the patient then received an additional two courses of combination therapy. This patient has lived without tumor recurrence for 10 years.Patient 1 was an 83-year-old Chinese woman with a 2-year history of an asymptomatic ulcerated mass on her left cheek. Physical examination in March 2008 revealed a 4 × 3.5 cm crater-like mass with central ulceration on her left cheek . Skin biPatient 2 was a 97-year-old Chinese woman with a 1-year history of a painful mass on her left cheek. Physical examination in November 2015 demonstrated the presence of a 2 × 2 cm mass with an irregular edge and signs of erosion on her left cheek . Skin biPatient 3 was a 76-year-old Chinese woman with a 1-year history of a large mass on her right ankle that had progressively increased in size. Physical examination in September 2012 revealed a 4 × 4 cm mass with erosion on her right ankle . Skin biA notable finding of this case report was that combination therapy achieved CSCC regression in all three patients within 2–6 months after the first administered dose of acitretin and clarithromycin. Furthermore, two of the patients exhibited complete remission of the tumor without scaring, thereby avoiding the need for surgery, while the other patient was successfully treated by tumor excision after combination therapy. Importantly, all three patients remained free of recurrence during a follow-up of 3–10 years. In addition, all three patients tolerated the treatment and experienced only minimal adverse effects (pseudotumor cerebri and mucocutaneous dryness) that resolved after discontinuation of acitretin. To our knowledge, this is the first study to report that combination therapy with acitretin and clarithromycin may be an effective treatment for CSCC.The majority of CSCCs can be successfully eradicated by surgical excision with post-operative margin assessment or microscopically controlled surgery, although the cosmetic results are not always satisfactory. When patients cannot or refuse to undergo surgery, alternative treatment options include radiotherapy, intralesional chemotherapy and photodynamic therapy. Radiotherapy is a potentially curative treatment in cases of early-stage CSCC where resection would cause substantial cosmetic or functional deficits. However, radiotherapy alone has limited effectiveness as a treatment for advanced CSCC and is associated with adverse effects. Anti-PD-1 antibodies are considered a first-line systemic treatment for patients with metastatic or locally advanced CSCC who are not candidates for curative surgery or radiotherapy, although its use in elderly patients is limited by the toxicity of this chemotherapeutic agent , 12. RecRetinoids are established drugs and have been shown to reduce the development of non-melanoma skin cancer in high risk individuals. Previous randomized controlled trials have demonstrated that systemic retinoids can prevent new primary CSCC or new precancerous lesions in immunocompromised patients after transplantation . TypicalAcitretin monotherapy is generally considered to have a low efficacy and is associated with dose-dependent side effects such as mucocutaneous dryness, hair loss, elevated triglycerides, transient elevations in liver function tests and (with long-term treatment) diffuse idiopathic skeletal hyperostosis. In view of the above issues, the patients in this case report were also administered clarithromycin, a macrolide antibiotic that has been reported to enhance the activity of conventional chemotherapy agents in cell lines and animal experiments , 9. FurtCombination therapy with acitretin and clarithromycin was well-tolerated by all three patients in this study. We summarized the common characteristics of the three patients in our study which may somehow contributed to the successful treatment outcomes. We found that all of them were female older than 75 and refused large-scale surgical treatment. The pathological diagnoses were poorly to well-differentiated invasive SCC. Their tumors were all single and without any metastases. We believe that elderly patients with slow tumor growth respond well to drug treatment and this can help them to avoid the risks associated with surgery. For young patients with multiple or larger tumors, the drug treatment can also be used to reduce the size of the tumor, furthermore it will be beneficial to surgical resection. The only adverse effects reported were pseudotumor cerebri and mucocutaneous dryness, which are known side-effects of acitretin. Therefore, we suggest that the combination of acitretin and clarithromycin may represent an effective and safe therapy for CSCC.As far as we are aware, this is the first study to report that combination therapy with acitretin and clarithromycin may be an effective and safe treatment for CSCC. Additional studies are required to further assess the efficacy and safety of combination therapy with acitretin and clarithromycin in the treatment of CSCC in larger populations and to investigate the underlying molecular mechanisms.The original contributions generated in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by The study was approved by the Medical Technology Clinical Application Ethics Committee of the First Affiliated Hospital of Fujian (No. [2015]084). The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.YZha: conception and design. CJ: administrative support. YZha, YZhu, and HW: provision of study materials or patients. CJ and YZhu: data analysis and interpretation. All authors: collection, assembly of data, manuscript writing, and final approval of manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


There is dearth of information on the timeliness of antenatal care (ANC) uptake. This study aimed to determine the timely ANC uptake by a medically trained provider (MTP) as per the World Health Organization (WHO) recommendations and the country guideline.Cross-sectional survey was done with 2,731 women having livebirth outcome in last one year in Dinajpur, Nilphamari and Rajshahi districts, Bangladesh from August-November,2016.st trimester by a MTP. As per 4 schedule visits by the WHO FANC model and the country guideline 8%(203) and 20%(543) women respectively received the first 2 timely ANC by a MTP; where only 1%(32) and 3%(72) received the first 3 visits timely and 0.6%(17) and 1%(29) received all the four timely visits. Factors significantly associated with the first two timely visits are: 10 or above years of schooling of women [adj. OR 2.13 ] and their husbands [adj. OR 2.40 ], women’s employment [adj. OR 2.32 ], urban residential status [adj. OR 3.49 ] and exposure to mass media [adj. OR 1.58 ] at 95% confidence interval. According to the 2016 WHO ANC model, only 1.5%(40) women could comply with the first two ANC contacts timely by a MTP and no one could comply with all the timely 8 contacts.About 82% women received at least one ANC from a MTP. Overall, 78% women received 4 or more ANCs by any provider and 43%(1168) from a MTP. Only 14%(378) women received their first ANC at the 1Despite high coverage of ANC utilization, timely ANC visit is low as per both the WHO recommendations and the country guideline. For better understanding, further studies on the timeliness of ANC coverage are required to design feasible intervention for improving maternal and child health. Approximately, 300,000 women die annually from pregnancy or childbirth-related complications around the world and almost all of these deaths occur in low-resource settings, and most of these deaths are preventable , 2. The High coverage of quality Antenatal Care (ANC) can play a crucial role to decrease maternal and child mortality rates and achieve national and global targets related to maternal and child health –7. StudiAs per the previous World Health Organization’s (WHO) recommended Focused Antenatal Care (FANC) Model; under normal circumstances, a pregnant woman should have at least four ANC visits . RecentlGlobally, the coverage of early ANC visit within 14 weeks is reported to increase from 40.9% to 58.6% from the year 1990 to 2013 . HoweverAccording to the Bangladesh Demographic and Health Surveys (BDHS) the trend of ANC coverage by a medically trained provider (MTP) is increasing , 22–24. Despite having a rise in ANC coverage in Bangladesh, it stands among the top ten countries those are contributing nearly 60% of global maternal mortality , 27. MatAccording to the BDHS-2017-18, less than 18% women received quality ANC care. Quality care is defined as receiving four or more antenatal visits, with at least one visit from a MTP and the components include measurement of weight and blood pressure, testing of blood and urine and receipt the information on potential danger signs during pregnancy . In addiIt was a community-based cross-sectional study conducted in both rural and urban areas. We had two study sites in rural areas and one in urban area from 3 northern districts of Bangladesh. Rural areas were Chirirbandar, a sub-district from Dinajpur and Saidpur, a sub-district from Nilphamari in Rangpur division and the urban area was Rajshahi City Corporation from Rajshahi division. According to the Population and Housing Census, the total population of Chirirbandar was 292,500, of which 146,619 were males, and 145,881 were female . For SaiWe applied two stages cluster sampling to select study participants in the study area. We considered some socio-demographic characteristics including age, years of schooling, occupation, religion, gravida and place of residence of study participants in the sampling frame to cover a range of information on similar issues from a variety of study participants. We considered government and non-government ‘Community-Based Health Workers’ (CBHW) catchment area as a cluster. We maintained similar population coverage for cluster selection. In Chirirbandar, we considered government CBHW’s catchment area, and for Saidpur and Rajshahi, we considered non-government CBHW’s areas as our clusters. In the first stage, we randomly selected one sub-district from two rural districts each and 10 wards (lowest administrative unit of city area) from the city corporation area. Then, we randomly selected 6 clusters (CBHW’s catchment area) out of 12 in Chirirbandar of Dinajpur district, 6 clusters out of 12 clusters in Saidpur of Nilphamari district and 6 clusters from 10 clusters in Rajshahi city area.To recruit study participants, we applied the Expanded Program of Immunization method, which is a popular spatial sampling method named as the EPI method. We selected the starting point to start data collection in the selected cluster using the EPI method. We determined the midpoint of each cluster in consultation with the community people. To ensure the randomization process in interviewing eligible participants, we spun a bottle at the midpoint of each cluster to identify the direction from where we started searching study participants , 41. IntIn each study area, around 900 women were interviewed, and finally, we completed 2731 interviews from the 3 study areas. Followings were the inclusion criteria for the study enrolment: (1) the woman had a live birth outcome in the last one year prior to interview (2) the woman passed 28 or more days after last delivery (3) the woman could hear, see and speak (4) the woman had permanent residence in the study area.We conducted this survey from August to November 2016. An expert research team from the International Centre for Diarrhoeal Disease Research, Bangladesh  was involved in preparing a survey tool based on the research question and objectives of the study. Study investigators trained all the data collectors on the data collection tool. After that, a field test was done as pre-testing to check the feasibility of the survey tool in the real field. We checked the consistency of the survey tool and incorporated the feedback into the final version after pre-testing. The survey was administered through face-to-face interviews with eligible women.An efficient team with an experienced team leader was closely involved with the data collection for ensuring the quality data. The team leader checked the completeness of every interview on the spot after the data collection through day to day supervision. Furthermore, a project research physician (PRP) and a research investigator (RI) coordinated all the data collection teams and team leaders on a daily and weekly basis for ensuring quality data and completeness of the interview. Re-interview was done by the team leaders, PRP and RI in a significant amount to check the accuracy and validity of data. At the same time, a database template was designed by an expert programmer of the Maternal and Child Health Division (MCHD), icddr,b to enter all the data online. Dot net (Version-10) software was used for data template design as appropriate . The datThis study had no more than minimal risk to study participants. We obtained written informed consent from each of the participants prior to the interviews. We received approval from the Institutional Review Board (IRB) of icddr,b before data collection in the field. All the participants were married, and there was no need for obtaining consent for the minors from the guardian or parents as per IRB.All the information given by the mothers were self-reported, however, we found 27% had a pregnancy registration card  and we checked their documents for relevance. Rest of the women who did not have a pregnancy registration card, we applied the probing technique to get the actual information from them. We determined the timely ANC coverage with regards to the WHO and the country guidelines. The primary outcome variable was the first two timely ANC visits by a skilled provider as per the WHO FANC model. Our country guideline resembles with the WHO FANC model and did not yet adopt the WHO 2016 model . We consnd ANC visit between 24–26 weeks, the 3rd ANC visit at 32nd week, and the 4th ANC Visit between 36–38 weeks of gestation [To analyze the timely ANC visits, we followed the criteria suggested by the two WHO models and country guideline. According to “the WHO FANC model”, the timely ANC visits refer to the 1st ANC visit between 8–12 weeks of pregnancy, the 2estation .th week, the 3rd contact at 26th week, the 4th at 30th week, the 5th at 34th week, the 6th at 36th week, the 7th at 38th week and the 8th contact at 40th week [nd ANC visit between 24–28 weeks, the 3rd ANC visit at 32nd week, and the 4th ANC Visit at 36th week of gestation [The timely ANC visits recommended by “the WHO 2016 ANC model”, refers to 1st contact within 12 weeks, the 2nd contact at 200th week . Like thst contact whereas the WHO FANC Model recommended the 1st visit between 8–12 weeks of gestation and the Bangladesh guideline recommended the 1st visit within 16 weeks of gestation.There is a slight difference among the 2016 WHO ANC Model, the WHO FANC Model and the Bangladesh guideline for recommending the1st timing of ANC. The 2016 WHO ANC Model recommended within 12 weeks of gestation for the 1st, 2nd and 4th visits in ranges and 3rd visit on the exact time of gestational age. The country guideline suggests timing for 1st and 2nd visits in ranges and 3rd and 4th visits on the exact time of gestational age. The recent WHO 2016 ANC model suggests only first contact in range of gestational weeks and remaining 7 contacts on the exact timing of gestational weeks.All the guidelines mentioned about the exact timing and ranges depending on gestational age. The WHO FANC model suggests timing for 1We considered Anderson and Newman’s framework of health services utilization to select the covariates that are associated with ANC utilization. This framework consists of three individual determinants- i. Predisposing ii. Enabling iii. Illness level , 45. We We performed statistical analysis using the statistical software package STATA version 13.1 . To iden“st ANC visit by gestational weeks from a skilled, unskilled and any provider. About one-fifth of the women (21%) received their 1st ANC within 12 weeks from any provider whereas 14% of them received from a skilled provider. The highest number of women (29%) received their 1st ANC between 13–16 weeks by any provider, whereas half of them (16%) received from a skilled provider.““th week from any provider and near to one-third (27%) from a skilled provider. More than one-third of the women (39%) received contact 3 at 26th week and contact 5 at 34th week by any provider whereas one-fourth women received those contacts from a skilled provider. In line with the 2016 WHO ANC model, less than one-fifth of the women (16%) did not have any timely ANC contact from any provider and almost two-thirds of the women (63%) received at least one timing contact from a skilled provider. Only a few women (0.15%) received the timely first five contacts as per the 2016 ANC model from MTP. None of the women received all the 8 timely ANC contact either by a MTP or by any provider. Urban women were more likely to receive timely ANC contacts than those of rural women. There were significant differences in receiving timely contacts by any provider except contact 2 and 6; and by a skilled provider except contact 2 based on place of residence.“““This study shows that almost all the women from both rural and urban sites received at least one ANC and more than three-fourth of the women received 4 or more ANC visits by any provider in their last pregnancy. Less than one-fifth of the women received 8 or more contacts by any provider whereas only 4% women received at least 8 contacts by MTPs. However, this uptake rate significantly differs between urban and rural women.st ANC visit within recommended time is mostly delayed. There is very little difference in terms of receiving the timely 1st ANC visit by both MTPs and any provider between the two WHO models. There is slight difference (13% Vs 14% by MTP and 20% Vs 21% by any provider) between the two models on the 1st ANC uptake and that is due to different recommended timings. The FANC model recommends first ANC between 8–12 weeks while the 2016 ANC model suggests to have the first ANC contact within 12 weeks. Therefore, when ANCs are received before 8 weeks, have been considered in the 2016 ANC model and was excluded in the FANC model. However, as per country guideline, the number of the 1st ANC uptake within 16 weeks of gestation by both MTPs and any provider was two times higher than the both WHO models. As Bangladeshi country guideline recommends an elaborated time range for the initiation of ANC, it kindles curiosity among all on the difference. Similar to our findings, three more studies were done in Bangladesh suggest that the uptake of first ANC is substantially delayed and another study revealed that the reasons could be maternal age, women’s education, residence, wealth index, pregnancy intention status, child’s birth order, and wanting more children [st ANC for few weeks thinking that pregnancy may terminate (abortion may occur) at an earlier stage, so they wait until 3 to 4 months of the pregnancy to report or visit healthcare centre. May be considering the cultural context, the national guideline adopted 1st ANC by 16 weeks. Although there is no evidence, however, we are assuming that socio-cultural factors associated with delayed ANC care seeking might have been reflected on our national guideline and thus on the recommendations of the time ranges.According to the WHO FANC and the 2016 ANC model, the practice of receiving the 1children , 48, 49.Though there are differences in timing, according to the WHO FANC model and the country guideline the highest proportion of the women received the timely ANC visit 2 by MTPs. Whereas according to the 2016 WHO ANC model, the highest proportion of the women complied with the contact 4 by MTPs. The country guideline mostly matches with the WHO FANC model but the 2016 WHO ANC model focused on the ANC contacts on exact weeks rather than considering ranges of weeks except ‘contact 1’ thus influences the variation of ANC uptake.As per the WHO FANC model and the country guideline, coverage of all four timely ANC visits were extremely low and no woman could follow all the 8 timely contacts recommended by the 2016 WHO ANC model regardless of the providers. However, completing the 8 contacts is not applicable if women delivered babies before 38 weeks.Because of the low observations for the all timely visits in both of the WHO models, the regression analysis of this study was limited to only the first two timely visits by a skilled provider as per the WHO FANC model. The regression analysis shows that women and their husbands with more years of schooling, employment, and living in the urban area were more likely to have the 1st two timely visits by MTPs.Regarding the socio-demographic characteristics like marriage, family planning, and childbearing, Northern Bangladesh shows some variations with other Northeast and Southeast regions of Bangladesh. Early marriage (marriage before 18 years) among Northern Bangladeshi women (Rajshahi: 70% and Rangpur: 67%) is relatively higher than other parts of Bangladesh; therefore the prevalence of teenage childbearing status is also higher (Rajshahi: 33 and Rangpur: 32 vsSylhet: 14 and Chattogram: 27). However, Total Fertility Rate is lower in Northern part (Rajshahi and Rangpur: 2.1) than Northeast (Sylhet: 2.6) and Southeast (Chattogram: 2.5) regions. Regarding the modern contraceptive usage and unmet need for family planning, Rajshahi  and Rangpur  stand in better position than Sylhet  and Chattogram  divisions .The BDHS 2017–18 data shows that the four or more ANC coverage raised to 47% from 31% in 2014. From the BDHS data for the Northern region, we found almost similar results with our study in terms of any ANC coverage by a MTP (BDHS: Rajshahi-84.5% and Rangpur-74.6% Vs this study-82%). The BDHS does not provide regional variation for number of ANCs, so, we couldn’t compare ANC coverage by numbers with BDHS. In addition to that, BDHS also does not present ANC coverage for 8 contacts . Our stuIn terms of four or more ANC coverage by any provider, a noticeable regional variation was observed for several South-Asian countries , 54–60. st ANC uptake by gestational age, this study found that only one-fifth of the women availed their 1st ANC in the 1st trimester (within 12 weeks) and more than one-fourth of the women received their 1st ANC during 13–16 gestational weeks by any provider. Another Bangladeshi study conducted in Netrokona district found ANC uptake by a formal provider  in the 1st trimester is 18% [st ANC by any provider and 14% received by MTPs [st ANC uptake [st ANC uptake in the 1st trimester, findings from several studies conducted in India showed the regional variation [st ANC in the 1st trimester, whereas in Andhra Pradesh more than three-fourth of the women and in eight other EAG states (Empowered Action Group) less than one-fifth of the women took the 1st ANC uptake in the first trimester [Regarding the 1r is 18% . Althoug by MTPs . We assuC uptake , 56–60. ariation , 60. Indrimester , 60. Howrimester .Though we found Afghanistan’s four or more ANC uptake is lower than our study, but surprisingly; Afghanistan’s 1st ANC uptake in the 1st trimester was a bit higher than that of ours while Pakistan and Nepal showed more than double ANC uptake rate than our findings , 58, 59.Studies show that the utilization of ANC in developing countries depends on many different factors –66. DiffSimilar to other low and middle-income countries (LMIC), Bangladesh is also improving its ANC coverage. The recommended 4 ANC visits was in a view of cost-effective model and result of extensive research, further, WHO recommended 8 ANC contacts in 2016 to expedite the improvement of Maternal and Child Health related status , 72. HowMounting all findings together from this and previous studies, we found that urban women can avail more ANC services than rural women although the ANC services are free of cost in government facilities everywhere in Bangladesh , 75. BasEven after conducting our study in high performing areas in terms of ANC coverage, extremely low prevalence of timely ANC uptake was observed maintaining the WHO and country guidelines. We can assume further worst-case scenario for low performing areas. Although we focused to discuss about ANC uptake by skilled provider in our result mostly but we found that many other national and also global studies we discussed in our paper tend to discuss the ANC uptake by any provider and used slightly different definition of skilled provider than country guideline , 68–70. Study team strictly maintained the quality of data collection in the field with close monitoring and supervision. The data derived from participants were rigorously rechecked and re-interviewed by team supervisors including a physician to minimize the scope of inaccuracy. We also checked relevant documents  during our data collection to minimize the errors. Since this study was conducted only in part with higher ANC coverage, findings of this study hence do not represent Bangladesh uniformly. Again, the analysis was done depending on self-reported information without having a robust surveillance system; therefore, the scope of over or under-reporting may exist. Because of self-reported data, number and timing of ANC visits can be varied. According to all three guidelines, there are variations on the timing of ANC visits by exact week (e.g. 30th week) and range of weeks (e.g. 8–12 weeks); and result from range weeks will vary less but results from exact weeks may vary little bit higher. More to add, we did not explore important potential exposure variables such as household income, awareness about maternity care, cost of service, availability of healthcare services and proximity to the health facility which might have served as confounders and affected the result and the interpretation of the findings.The coverage of ANC visits is quite high but the timeliness of ANC visits is very low as per both WHO models and country guideline. Initiation for the first ANC visit is also highly delayed. Government and non-government maternal health programs should focus on ensuring timely ANC visits. Ensuring at least 4 timely visits may help to make a way forward for Bangladesh endorsing 8 ANC contacts in the near future that is recommended by the recent 2016 ANC model. We suggest policy makers to promote education, women’s employment and health education through mass media as well as to ensure universal maternal healthcare coverage. Understanding the significance of timely ANC visits we further suggest to carry out more parallel studies both in countrywide and regional perspective putting emphasis on the feasibility of 8 contacts. The findings of this study will help the program and policy makers to design interventions to improve antenatal care coverage maintaining timeliness and thus reduce maternal and child mortality across Bangladesh.S1 File(DOCX)Click here for additional data file.S1 Data(DTA)Click here for additional data file.


Arabidopsis root tip. We tested the model predictions using the DII-VENUS auxin response reporter, comparing the predicted and observed DII-VENUS distributions using genetic and chemical perturbations designed to affect both carrier-mediated and plasmodesmatal auxin fluxes. The model revealed that carrier-mediated transport alone cannot explain the experimentally determined auxin distribution in the root tip. In contrast, a composite model that incorporates both carrier-mediated and plasmodesmatal auxin fluxes re-capitulates the root-tip auxin distribution. We found that auxin fluxes through plasmodesmata enable auxin reflux and increase total root-tip auxin. We conclude that auxin fluxes through plasmodesmata modify the auxin distribution created by efflux and influx carriers.Auxin is a key signal regulating plant growth and development. It is well established that auxin dynamics depend on the spatial distribution of efflux and influx carriers on the cell membranes. In this study, we employ a systems approach to characterise an alternative symplastic pathway for auxin mobilisation via plasmodesmata, which function as intercellular pores linking the cytoplasm of adjacent cells. To investigate the role of plasmodesmata in auxin patterning, we developed a multicellular model of the  Highlighted Article: A multicellular model of auxin transport in the Arabidopsis root tip reveals the importance of intercellular plasmodesmata in maintaining auxin pattern and fluxes. The plant hormone auxin regulates plant growth and plays a key role in many developmental responses . Within Auxin is transported through plant tissues both by passive diffusion and by specialized proteins located on cell membranes (which are often referred to as ‘carriers’). The plasma membrane-localised PIN proteins facilitate efflux of anionic auxin from the cell cytoplasm to the apoplast and are typically polar localised within the root tip . AUX1/LANumerous computational models have focused on how carrier-mediated auxin transport leads to the root-tip auxin dynamics  and haveIn addition to auxin transport, local auxin metabolism also influences the root-tip auxin distribution and the resulting root phenotypes, and can be regulated by other hormones and environmental conditions . Key auxFig. S1), , . We previg. S1), . In thisWe hypothesised that the discrepancy between the predicted and observed DII-VENUS distributions could be caused by the presence of an additional transport component such as diffusion through plasmodesmata, which are narrow pores directly linking the cytoplasm of adjacent plant cells . One stuFigs S2, S3; Materials and Methods). In addition, weak background efflux carriers were included on all cell membranes to account for non-polar PINs and the ABCB transporters. We specified low auxin production and degradation rates within every cell and higher auxin production in the quiescent centre (QC) and columella initials and lateral root cap (LRC), reflecting the observed distributions of auxin biosynthesis enzymes , which were previously derived and parameterised using auxin-dose-response data in Table S2) the steady state of the system of ODEs was computed directly using a linear system solver in Python to give the predicted root-tip auxin and DII-VENUS distributions. See Materials and Methods and supplementary Materials and Methods for further details.With these data and model assumptions, we generated a system of linear ordinary differential equations (ODEs) for the auxin concentration within each cell and cell-wall compartment, which contain terms representing passive diffusion of protonated auxin across cell membranes, carrier-mediated transport of anionic auxin across cell membranes, passive auxin diffusion within the cell wall and auxin synthesis and degradation. This system of ODEs was augmented by ODEs representing the network of interactions through which auxin degrades DII-VENUS  showed that the AUX1 influx carriers have a significant effect on the auxin distribution and appear to determine which tissues have high auxin ; however, the predicted DII-VENUS distribution again showed greater contrast than was observed experimentally .We also quantitatively compared the predicted and observed DII-VENUS distributions in an Fig. S6A). However, with PIN2 removed, the predicted auxin distribution  is similar to that observed in wild type except in the columella region where we predict higher auxin concentrations in pin2  pattern that is perturbed in pin2, is consistent with our previous suggestion that AUX1 and LAX determine the tissues with high auxin whereas PINs mediate the fluxes within these tissues . Using the corresponding root-tip template and removing PIN2 from our simulations   M, suggesld type) K. As a rthe data L.pin2 mutant, PIN1 is ectopically expressed and localised on the shootward-facing membranes within the meristem, following the PIN2 expression pattern, thus partially restoring the wild-type PIN efflux carrier pattern , with slightly less auxin in the division zone and slightly more in the elongation zone with ectopic PIN1, suggesting that the observed ectopic PIN1 is not sufficient to restore the wild-type auxin dynamics in the pin2 mutant.Previous experimental studies have shown that in the  pattern . To testpin2 .We initially hypothesised that increasing the level of the non-polar background efflux (which represents the auxin fluxes mediated by the non-polar PIN and ABCB membrane proteins) might enable auxin to leave the outer AUX1-expressing cells to move to the inner cell layers, which would improve agreement between the predicted and observed DII-VENUS distributions. To test this hypothesis, we increased the non-polar background permeability, but found only a small improvement in the agreement between predicted and observed DII-VENUS distributions in both wild type and pin2, using permeability parameter values set to both half and double their original value for PIN efflux , AUX1 influx  and LAX influx . In each case, we predicted minor differences in DII-VENUS levels; however, the overall pattern remains the same. In all cases considered, DII-VENUS was predicted to be low in the elongating epidermis and cortex in pin2, in contrast to the data, and we therefore concluded that changing the permeability parameter values does not enable the model to agree with the data.We also considered whether changing the values of the permeabilities associated with the carriers would improve the agreement between the predictions and data, as the values of these parameters have not been well characterised . To testWe concluded that our current model is unable to reproduce the DII-VENUS data and that carrier-mediated auxin transport alone does not appear to account for root-tip auxin distribution.Experimental studies have detected significant plasmodesmatal fluxes within the root tip  and haveplas. Between adjacent cells, we specified the plasmodesmatal density using the detailed electron microscopy data of Table S1). These data suggest that the plasmodesmatal density is high between adjacent cells in each tissue layer (5.42-12.58 plasmodesmata per µm2), but low between adjacent cells of different tissue layers (2.33-3.08 plasmodesmata per µm2). We estimated a value of Pplas=0.8 μm3 s−1 by dividing the permeability of 8.0 μm s−1 recorded by Arabidopsis root stele by the plasmodesmatal density of 9.92 μm−2 measured in the same tissue by plas, having units of μm3 s−1 .Plasmodesmata enable passive auxin diffusion between the symplast of adjacent cells, thus enabling auxin fluxes from cells of high concentration to those of low concentration. We introduced this into the model by incorporating additional terms to the system of ODEs, prescribing the plasmodesmatal auxin flux between adjacent cells to be proportional to the concentration difference in auxin between each cell's cytoplasm. In these plasmodesmatal flux terms, the proportionality constant is equal to the  density of plasmodesmata multiplied by a constant permeability per plasmodesmata, Ppin2 and aux1 . In wild type and pin2, plasmodesmata allow auxin to diffuse from the cells in the outer root tissue layers, where auxin concentrations are high, into the inner tissue layers, where auxin concentrations are lower. Thus, the presence of plasmodesmata increases the predicted auxin concentration in the meristematic epidermis and cortex underlying the LRC, for example, in agreement with the observed DII-VENUS distributions  and that the introduction of plasmodesmata improves agreement with the DII-VENUS experimental data in both cases .We also checked whether ectopic PIN1 in aux1 and pin2 , many of these are not known precisely; in particular, the auxin biosynthesis and degradation rates and the permeabilities associated with each of the membrane proteins have not been well characterised. We therefore performed a parameter survey to assess whether the values of each of these parameters affect our results and conclusions. We found that including plasmodesmatal auxin fluxes improves agreement between the model predictions and data for wild type, aux1 and pin2 for wide ranges of each of these parameter values .Although values for the model parameters have been suggested in the literature . Simulating NPA treatment in our model required us to reduce both PIN permeability and the background permeability by a fixed  amount. Our model without plasmodesmata predicted that reducing the PIN and background permeabilities makes little difference to either the peak level of DII-VENUS or the spatial pattern until the reduction in permeability is at or near 100% (effectively eliminating efflux entirely) , whereas the model predictions with plasmodesmata show a gradual reduction in both the peak level of DII-VENUS and in the sharpness of the spatial pattern . Using our fitness measure described above, we see that whatever the true level of reduction in auxi2n efflux efficacy following our NPA treatment, the model with the plasmodesmata is an improvement on the model without plasmodesmata .To test the role of plasmodesmata further, we also considered seedlings treated with the auxin efflux inhibitor naphthylphthalamic acid (NPA) . InteresIn summary, we found that introducing plasmodesmatal auxin fluxes improves agreement between the model predictions and data in all cases considered. We concluded that plasmodesmatal auxin fluxes are essential to re-capitulate the experimentally derived auxin distribution.The model revealed that the presence of plasmodesmata enables auxin to diffuse passively from the shootward auxin streams through the root's outer layers (where auxin concentrations are high) to the rootward auxin streams in the inner layers C. For explas , so as a result the total auxin in the root tip decreases.We observed that the model appears to predict that the root-tip auxin concentrations are higher when plasmodesmata are included B and 2B.plas K. For alFig S20). Similar effects are observed in the pin2 and aux1 models . We concluded that differences in plasmodesmatal density . With PIN carriers alone, when the upstream auxin concentration is increased, a wave of this higher auxin concentration propagates through the cell file . These single-file simulations revealed that the high plasmodesmatal density between adjacent cells within each tissue layer enables plasmodesmatal diffusion to modify the shootward and rootward auxin fluxes created by the PIN carriers (without affecting the effective PIN-mediated auxin velocity through the cell file).To assess how plasmodesmatal density affects the auxin fluxes through the individual tissue layers, we simulated the auxin dynamics through a file of single cells, with polar PIN carriers on the downstream cell membranes , whereas in aux1  there appears to be a less well-defined auxin maximum around the QC .We also applied the 0.6 mM HWe next genetically manipulated plasmodesmatal permeability. Plasmodesmatal permeability is modified by callose deposition , and keyGSL8, which is highly expressed in root tips  after an auxin dose, suggesting that plasmodesmatal permeability does not appear to be affected by auxin levels in the root . We therefore employed a dexamethasone (DEX)-inducible GSL8 anti-miRNA DII-VENUS line to simultaneously downregulate callose deposition and monitor auxin levels. We observed a more uniform DII-VENUS distribution, which is in good agreement with the model predictions  . In contrast, the mock-treated control gsl8 root (in which GSL8 is still expressed) exhibits higher DII-VENUS, both experimentally and in the model, than the DEX treated root , but also predict the flux pattern within the root tip, which cannot be detected directly experimentally. We found that plasmodesmata enable fluxes between adjacent tissue layers by allowing auxin to move between the transport streams created by the polar PIN proteins. Plasmodesmatal auxin fluxes thus create a reflux loop  at the root tip was measured recently  and the In addition to auxin, plasmodesmata are thought to be conduits for many signals, such as microRNAs, transcription factors, water and nutrients ; for exaMulticellular root tip geometries were obtained from the confocal images using SurfaceProject and CellSeT .supplementary Materials and Methods for a full model definition.These data, along with the assignation of auxin influx and efflux carriers to specific cell membranes based on neighbouring cell types, were used to generate a system of linear ODEs for the auxin concentration within each cell and cell-wall compartment and DII-VENUS within each cell. Along with small production and degradation terms in every cell, there was a fixed, non-zero boundary condition for auxin in the stele at the shootward end of the tissue, representing a constant supply of auxin from the shoot. The steady state of the ODEs was computed directly using a linear system solver in Python. See Fig. S3. PIN1 was specified to be on the rootward-facing membranes in the endodermis and stele. PIN2 was specified to be on the shootward-facing membranes in the LRC, elongation zone and distal-meristem epidermis and elongation-zone cortex, and on the rootward-facing membranes of the meristematic cortex. PIN3 was specified to be on the rootward-facing membranes of the stele and endodermis, the inward-facing lateral membranes of the endodermis and all faces of the columella initials and S1 and S2 tiers; PIN4 to be on rootward-facing membranes in the proximal meristematic cells of the epidermis, cortex, endodermis and stele and on all faces of cells in the QC, columella initials and S1 and S2 tiers. PIN7 was specified to be on the rootward-facing membranes in the stele. We specified AUX1 to be present in the LRC, elongation zone epidermis and cortex (with expression in the cortex first appearing in a more shootward position than in the epidermis), and S1, S2 and S3 tiers of the columella; LAX2 to be present in the QC, columella initials, and rootward half of the meristematic stele; and LAX3 to be present only in the S2 tier of the columella.The prescribed carrier distributions are shown in −2 s−1). The Arabidopsis ecotype Columbia (Col-0) was used as the wild type in all experiments.Seeds were surface sterilised with 50% (vol/vol) hypochlorous acid for 5 min and then washed three times with sterile deionised water. Plant seeds were plated on 0.5 strength Murashige and Skoog medium , at pH 5.8 and solidified with 1% plant agar (Duchefa). Seeds were stratified at 4°C for 48 h in the dark to synchronise germination, and then incubated vertically in a culture room under 12 h light at 22°C and 12 h dark at 22°C . Scanning settings used for one experiment were optimised and kept unchanged throughout the experiments.Confocal microscopy was performed using a Leica SP8 confocal laser scanning microscope (Leica Microsystems). Cell walls were stained using propidium iodide (10 µg ml


COVID-19 presents challenges to the emergency care system that could lead to emergency department (ED) crowding. The Huddinge site at the Karolinska university hospital (KH) responded through a rapid transformation of inpatient care capacity together with changing working methods in the ED. The aim is to describe the KH response to the COVID-19 crisis, and how ED crowding, and important input, throughput and output factors for ED crowding developed at KH during a 30-day baseline period followed by the first 60 days of the COVID-19 outbreak in Stockholm Region.Different phases in the development of the crisis were described and identified retrospectively based on major events that changed the conditions for the ED. Results were presented for each phase separately. The outcome ED length of stay (ED LOS) was calculated with mean and 95% confidence intervals. Input, throughput, output and demographic factors were described using distributions, proportions and means. Pearson correlation between ED LOS and emergency ward occupancy by phase was estimated with 95% confidence interval.As new working methods were introduced between phase 2 and 3, ED LOS declined from mean (95% CI) 386 (373–399) minutes to 307 (297–317). Imaging proportion was reduced from 29 to 18% and admission rate increased from 34 to 43%. Correlation (95% CI) between emergency ward occupancy and ED LOS by phase was 0.94 (0.55–0.99).It is possible to avoid ED crowding, even during extreme and quickly changing conditions by leveraging previously known input, throughput and output factors. One key factor was the change in working methods in the ED with higher competence, less diagnostics and increased focus on rapid clinical admission decisions. Another important factor was the reduction in bed occupancy in emergency wards that enabled a timely admission to inpatient care. A key limitation was the retrospective study design. The COVID-19 outbreak presents large challenges to the emergency care system. The underlying SARS-CoV-2 virus appears highly contagious to both staff and other patients and the resultant disease is potentially deadly, especially for older patients with underlying comorbidities such as active cancer, obesity, coronary artery disease, diabetes and chronic obstructive pulmonary disease –5 as patThere is a high risk that this increased workload could lead to ED crowding  that is The aim is to describe how the Huddinge site at the Karolinska university hospital (KH) responded to the COVID-19 crisis, and how ED crowding, and important input, throughput and output factors for ED crowding developed at KH during a 30-day baseline period followed by the first 60 days of the COVID-19 outbreak in Stockholm Region.This is a retrospective descriptive study of KH response to the COVID-19 crisis. In the study, the development of the crisis was divided into six different phases separated by five major events that changed the conditions for the ED . Throughput factors indicating the share of diagnostics and treatment performed in the ED were the proportion of patient visits that included medical imaging during the ED stay and the proportion of patient visits that resulted in an admission to inpatient care. Output factors were the bed occupancy level in the emergency wards and the number of care episodes at the emergency wards, together indicating the available capacity for the ED to admit patients to inpatient care without a finalized diagnosis. Another output factor was the total hospital bed capacity indicating the general ability to admit patients to inpatient care. To provide a view on this, the average number of staffed inpatient beds together with the average number of patients by type of ward, phase and date were presented. The wards were grouped in four categories: intensive care, emergency care, infection, and other wards (excluding pediatric and obstetric wards). Basic demographic information on the distribution of sex and age in intervals < 40, 40–59, 60–79 and 80+ were also presented.Data used in the study was based on statistics extracted from KH data warehouse. Crosstabs of data were delivered to the research team using Tableau Desktop 2018.3.0 and imported into RStudio 1.1.463 and R version 3.6.1 to create the table, the analysis of correlations and high-resolution graphs. ED LOS is measured from the first registration at the front desk or in the later stages in the tent outside the ED, until the patient leaves the ED. Bed occupancy is measured as the daily average of observations on number of staffed inpatient beds and number of admitted patients that is registered at each ward. The number of staffed inpatient beds is entered manually by each ward as soon as the status changes. The number of patients in each ward is updated automatically as patients are admitted and discharged in the electronic healthcare records. The Data warehouse stores information on beds and patients with 15 min resolution so each daily average is based on 96 observations.P-value and 95% confidence intervals where each phase represented a single observation were estimated between ED LOS and the key input, throughput and output factors. The input factor used was average visits per day, the throughput factor was proportion of imaging performed and the output factor was bed occupancy in the emergency wards. Development of staffed inpatient beds and average number of patients were visualized in line graphs by the type of ward, phase and date.For the outcome ED crowding, mean ED LOS was estimated with 95% confidence interval for each phase. Input, throughput and output factors previously known to impact ED crowding together with age and sex were described for each phase using distributions, means and proportions. Pearson correlations with In total there were 9754 ED visits during the 90-day study-period from February 1 to April 30. Three hundred ninety four visits (4%) were excluded due to missing triage priority while 13 visits (0%) were excluded due to missing information on sex or ED LOS. In total, 9347 visits (96%) were included.Visits per day were similar in the baseline phase 1 (118) and phase 2 (114) but declined in phase 3 (93). In phase 4 and 5 the number of visits increased to 103 and 123. This was mainly due to an increase in the number of EMS arrivals that reached a maximum of 75 in phase 5 compared to 43 in the baseline phase 1. The proportion of EMS arrivals increased to 49% in phase 4 and 61% in phase 5 compared to 37% in the baseline phase 1.In phase 3, the proportion of patients that had some kind of imaging performed during their ED visit dropped to 18% compared to 32% in the baseline phase 1 and then further to 15 and 11% in the following two phases. Admission to inpatient care developed in the opposite direction with an increase to 43% in phase 3, 48% in phase 4 and 51% in phase 5 compared to 33% in the baseline phase 1  ED LOS was 440 431–449) minutes at the baseline phase 1 and declined to 386 (373–399) in phase 2 and 307 (297–317) in phase 3. It then increased to 341 (321–360) in phase 4 and 351 (336–366) in phase 5 before again declining to 294 (287–302) minutes in the final phase 6. All phases following the implementation of new working methods in phase 3 had a statistically significant lower mean ED LOS than both the first two phases  0.71 (− 0.24–0.97) with P = 0.11 for mean ED LOS and proportion of imaging performed by phase.Pearson correlation (95% CI) between mean ED LOS and mean emergency ward bed occupancy for each phase was 0.94 (0.55–0.99) with 005 Fig. . ResultsIn this retrospective, descriptive study we analyzed the developments at KH during a 30-day baseline period followed by the first 60 days of the COVID-19 outbreak in Stockholm Region, Sweden. Analysis of 9347 ED visits separated in six phases show that KH was able to improve ED crowding despite the challenges posed by COVID-19. ED crowding is a complex, multivariable issue and multiple factors may have impacted this development. To structure the analysis, we used the conceptual input, throughput, output model for ED crowding introduced by Asplin et al.  The mainA key limitation was the study-design where the research group defined the cut-off points and the phases retrospectively. We aimed to define cut-off points as objectively as possible based on major events that impacted the conditions for the ED, but this may still have introduced bias. It was however not feasible to conduct a prospective intervention study as we were in a state of emergency, unable to predict how events would unfold and what measures that would be taken. Overall, the quality of the data is high as the study is based on standard reporting variables already existing in the data warehouse that is used daily. This is also a limitation as it narrows down the choice of proxy variables to use for the factors. One limitation is that ED LOS starts when the patient is registered and not when the patient arrives at the ED. When the arrival intensity is high, there may be additional waiting time before being registered. These situations are likely to be associated with ED crowding and extended ED LOS so high levels of ED LOS may be underestimated. Another limitation is that the number of staffed beds is based on manual input from the wards as capacity changes. This often leads to small delays as the priority is to first change the care capacity and then to report it in the system. This may have led to an underestimation of the intensive care capacity in the middle of phase 3. Generalizability may to some aspects be limited as there were some favorable conditions at KH. The hospital had a relatively large part of the staff engaged in research, education and specialized elective care that could be redeployed during the crisis. KH also have a large operating theatre with many physicians and nurses specialized in anesthesiology and perioperative care that could quickly be retrained to work in intensive care. In addition to this, the new operating theatre with pre- and postoperative wards were just recently inaugurated and had excellent capabilities to be transformed into intensive care wards for COVID-19 patients.It is possible to avoid ED crowding, even during extreme and quickly changing conditions by leveraging previously known input, throughput and output factors. One key factor was the change in working methods in the ED with higher competence, less diagnostics and increased focus on rapid clinical admission decisions. Another important factor was the reduction in bed occupancy in emergency wards that enabled a timely admission to inpatient care.