Lessons identified from initiating a thalassaemia programme in a conflict setting: a case study from northeast Syria.

BACKGROUND: Thalassaemia affects many families in Northeast Syria, an area devastated by over a decade of conflict which has significantly impacted their health system. People with thalassaemia require holistic multidisciplinary care for the clinical complications of thalassaemia. The risks of thalassaemia treatment include blood-borne viral infections secondary to unsafe transfusion, increased vulnerability to serious bacterial infection following splenectomy, and complications of both iron overload and iron chelation therapy. Médecins Sans Frontières (MSF) provided outpatient thalassaemia care programmes in northeast Syria between April 2017 October 2019 in a complex conflict context challenged by population displacement, the destruction of medical facilities, and periods of insecurity. METHODS: We performed a secondary descriptive analysis of the thalassaemia cohort data to describe basic clinical and demographic characteristics of the patient population. A desk review of internal and publicly available documents was supplemented by informal interviews with MSF staff to describe and analyse the programmatic approach. CASE DESCRIPTION: MSF delivered programmes with thalassaemia investigations, provision of blood transfusion, iron chelation therapy, and psychosocial support. Thalassemia programmes were novel for the organisation and operational learning took place alongside service implementation. Lessons were identified on equipment procurement and the requirements for the implementation of vital investigations (including ferritin testing), to inform clinical decision making. Lessons included the importance of supply planning for sufficient blood products to meet diverse clinical needs in a conflict area, so those with thalassaemia have continued access to blood products among the competing priorities. Iron chelation therapy met a large need in this cohort. Adapted protocols were implemented to balance social factors, hygiene considerations, toxicity, tolerability, and adherence to therapy. Wider service needs included considerations for family planning advice and services, continuity of care and patient access through decentralised services or laboratory access, psychosocial support, and improved data collection including quality of life measurements to understand the full impact of such programmes. CONCLUSIONS: Although this type of programming was not "routine" for the organisation, MSF demonstrated that life-sustaining thalassaemia care can be provided in complex conflict settings. International non-governmental organisations can consider this care possible in similar contexts.

Mumps-specific IgG, IgG subclasses and neutralization titres to the vaccine and outbreak mumps strains differ in vaccinated healthy controls, breakthrough mumps infection cases and naturally infected individuals.

BACKGROUND: Despite widespread use of the mumps vaccine resulting in significant reduction in the incidence of symptomatic mumps infection, large outbreaks continue to occur in highly vaccinated populations. OBJECTIVES: We examined the mumps-specific IgG, IgG subclasses and neutralization titres to the outbreak Genotype G5 and Jeryl Lynn vaccine (Genotype A) mumps strains. STUDY DESIGN: Sera from 207 individuals were classified into five distinct cohorts: healthy controls and mumps cases of 5-17 years and 18-25 years, and naturally infected individuals of 50+ years. Mumps specific IgG and IgG subclass levels were measured using modified ELISA assays with lysates and nucleoprotein antigens from both the mumps vaccine and circulating Genotype G5 strains. All sera were investigated for in vitro neutralizing antibody titres (GMT) using focus reduction neutralization assays. Data was analysed using the Kruskal-Wallis test and pairwise Wilcoxon tests. RESULTS: Mumps cases had higher mumps IgG levels compared to controls, to both the vaccine and outbreak strains, however levels decreased with age. Mumps IgG3 levels were significantly raised in mumps cases (p < 0.001). Neutralization titres were lower to the outbreak strain in all cohorts with titres markedly lower in the mumps cohorts compared to healthy controls. Mean GMT to the vaccine strain increased with age. The naturally infected group displayed the highest GMT to the JL vaccine and the lowest GMT to the outbreak strain. CONCLUSIONS: Antigenic differences between mumps vaccine strain and circulating mumps viruses decrease the cross-neutralization capacity of vaccine-induced antibodies which may play a role in breakthrough infection.

The psychological impact of childhood homelessness-a literature review.

In August 2019, 3848 children in Ireland were faced with emergency homelessness [1]. In recent years, lack of affordable housing, unemployment and shortage of rental properties have been the primary driving factors for the potentially devastating impact of familial homelessness in our society [1]. Our aim was to evaluate current knowledge on the psychological impact of homelessness in children. Using the PRISMA model, we performed a review of the currently available literature on the psychological impact of homelessness on children. This concept was explored under two different categories-'transgenerational' and 'new-onset homelessness'. Hidden homelessness was also explored. Our literature review revealed several psychological morbidities which were unique to children. This includes developmental and learning delays, behavioural difficulties and increased levels of anxiety and depression [66, 77, 40, 81, 42]. This has been demonstrated by poorer performance in school testing and increased levels of aggression. Anxiety in children within this cohort has been shown to peak at time of dispersion from their stable home environment [67]. Our study highlights violence, aggression and poor academic learning outcomes to be just some of the key findings in our review of homelessness in childhood, worldwide. Unfortunately, there has been minimum research to date on paediatric homelessness within the context of the Irish population. We anticipate this review to be the first chapter in a multipart series investigation to evaluate the psychological morbidity of paediatric homelessness within the Irish Society.

Physical illnesses associated with childhood homelessness: a literature review.

BACKGROUND: Childhood homelessness is a growing concern in Ireland [1] creating a paediatric subpopulation at increased risk of physical illnesses, many with life-long consequences [2]. AIM: Our aim was to identify and categorize the physical morbidities prevalent in homeless children. METHODS: A review of the English-language literature on physical morbidities affecting homeless children (defined as ≤ 18 years of age) published from 1999 to 2019 was conducted. RESULTS: Respiratory issues were the most commonly cited illnesses affecting homeless children, including asthma, upper respiratory tract infections, and chronic cough [3]. Homeless children were described as being at increased risk for contracting infectious diseases, with many studies placing emphasis on the risks of sexually transmitted infections (STIs) and HIV/AIDS transmission [4, 5]. Dermatologic concerns for this population comprised of scabies and head lice infestation, dermatitis, and abrasions [3, 6]. Malnutrition manifested as a range of physical morbidities, including childhood obesity [7], iron deficiency anemia [4], and stunted growth [8]. Studies demonstrated a higher prevalence of poor dental [7] and ocular health [9] in this population as well. Many articles also commented on the risk factors predisposing homeless children to these physical health concerns, which can broadly be categorized as limited access to health care, poor living conditions, and lack of education [3, 10]. CONCLUSION: This literature review summarized the physical illnesses prevalent among homeless children and the contributing factors leading to them. Gaps in the literature were also identified and included a dearth of studies focusing on younger children compared with adolescents. Further research into prevention and intervention programs for this vulnerable population is urgently needed.

Readmission to hospital following laparoscopic cholecystectomy: a meta-analysis.

BACKGROUND: Laparoscopic cholecystectomy (LC) is one of the most commonly performed surgical procedures. Despite this, patterns of readmission following LC are not well defined. This meta-analysis aimed to determine rates and predictors of readmission. METHODS: An ethically approved International Prospective Register of Systematic Reviews (PROSPERO)-registered meta-analysis was undertaken searching PubMed, Scopus, Web of Science and Cochrane Library databases from January 2013-June 2018 adhering to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. Published literature potentially suitable for data analysis was graded using methodological index for non-randomised studies (MINORS) criteria; papers scoring ≥ 16/24 for comparative and ≥ 10/16 for non-comparative studies were included. A meta-analysis of potential risk factors was performed by computing the odds ratio using Mantel-Haenszel method and fixed-effects model with 95% confidence intervals. RESULTS: Three thousand and eight hundred thirty-two articles were reduced to 44 studies qualifying for a final analysis of 1,573,715 laparoscopic cholecystectomies from 25 countries. Overall readmission rate was 3.3% (range: 0.0-11.7%); 52,628 readmissions out of 1,573,715 LCs. Surgical complications accounted for 76% of reported reasons for readmission, predominantly bile duct complications (33%), wound infection (17%) and nausea and vomiting (9%). Pain (15%) and cardiorespiratory complications (8%) account for the remainder. Obesity, single port LC and day case LC were not associated with increased rates. CONCLUSIONS: Pain, nausea and vomiting and surgical complications, particularly bile duct obstruction are the most common causes for readmission. Intra-operative cholangiography may reduce readmission rates. Causes for readmission were inconsistently reported throughout. The mean readmission rate of 3.3% may act as a quality benchmark for improving LC, and clearer reporting of reasons for readmission are required to advance care.

Inhibition of NGLY1 Inactivates the Transcription Factor Nrf1 and Potentiates Proteasome Inhibitor Cytotoxicity.

Proteasome inhibitors are used to treat blood cancers such as multiple myeloma (MM) and mantle cell lymphoma. The efficacy of these drugs is frequently undermined by acquired resistance. One mechanism of proteasome inhibitor resistance may involve the transcription factor Nuclear Factor, Erythroid 2 Like 1 (NFE2L1, also referred to as Nrf1), which responds to proteasome insufficiency or pharmacological inhibition by upregulating proteasome subunit gene expression. This "bounce-back" response is achieved through a unique mechanism. Nrf1 is constitutively translocated into the ER lumen, N-glycosylated, and then targeted for proteasomal degradation via the ER-associated degradation (ERAD) pathway. Proteasome inhibition leads to accumulation of cytosolic Nrf1, which is then processed to form the active transcription factor. Here we show that the cytosolic enzyme N-glycanase 1 (NGLY1, the human PNGase) is essential for Nrf1 activation in response to proteasome inhibition. Chemical or genetic disruption of NGLY1 activity results in the accumulation of misprocessed Nrf1 that is largely excluded from the nucleus. Under these conditions, Nrf1 is inactive in regulating proteasome subunit gene expression in response to proteasome inhibition. Through a small molecule screen, we identified a cell-active NGLY1 inhibitor that disrupts the processing and function of Nrf1. The compound potentiates the cytotoxicity of carfilzomib, a clinically used proteasome inhibitor, against MM and T cell-derived acute lymphoblastic leukemia (T-ALL) cell lines. Thus, NGLY1 inhibition prevents Nrf1 activation and represents a new therapeutic approach for cancers that depend on proteasome homeostasis.

Complementary Assays Reveal a Low Level of CA Associated with Viral Complexes in the Nuclei of HIV-1-Infected Cells.

UNLABELLED: During uncoating, the conical capsid of HIV disassembles by dissociation of the p24 capsid protein (CA). Uncoating is known to be required for HIV replication, but the mechanism is poorly defined. Here, we examined the timing and effect of two capsid binding drugs (PF74 and BI2) on infectivity and capsid integrity in HIV-1-infected cells. The virus remained susceptible to the action of PF74 and BI2 for hours after uncoating as defined in parallel drug addition and cyclosporine (CsA) washout assays to detect the kinetics of drug susceptibility and uncoating, respectively. Resistance mutations in CA decreased the potency of these compounds, demonstrating that CA is the target of drug action. However, neither drug altered capsid integrity in a fluorescence microscopy-based assay. These data suggest that PF74 and BI2 do not alter HIV-1 uncoating but rather affect a later step in viral replication. Because both drugs bind CA, we hypothesized that a residual amount of CA associates with the viral complex after the loss of the conical capsid to serve as a target for these drugs. Superresolution structured illumination microscopy (SIM) revealed that CA localized to viral complexes in the nuclei of infected cells. Using image quantification, we determined that viral complexes localized in the nucleus displayed a smaller amount of CA than complexes at the nuclear membrane, in the cytoplasm, or in controls. Collectively, these data suggest that a subset of CA remains associated with the viral complex after uncoating and that this residual CA is the target of PF74 and BI2. IMPORTANCE: The HIV-1 capsid is a target of interest for new antiviral therapies. This conical capsid is composed of monomers of the viral CA protein. During HIV-1 replication, the capsid must disassemble by a poorly defined process called uncoating. CA has also been implicated in later steps of replication, including nuclear import and integration. In this study, we used cell-based assays to examine the effect of two CA binding drugs (PF74 and BI2) on viral replication in infected cells. HIV-1 was susceptible to both drugs for hours after uncoating, suggesting that these drugs affect later steps of viral replication. High-resolution structured illumination microscopy (SIM) revealed that a subset of CA localized to viral complexes in the nuclei of cells. Collectively, these data suggest that a subset of CA remains associated with the viral complex after uncoating, which may facilitate later steps of viral replication and serve as a drug target.

The polysialyltransferases interact with sequences in two domains of the neural cell adhesion molecule to allow its polysialylation.

The neural cell adhesion molecule (NCAM) is the major substrate for the polysialyltransferases (polySTs), ST8SiaII/STX and ST8SiaIV/PST. The polysialylation of NCAM N-glycans decreases cell adhesion and alters signaling. Previous work demonstrated that the first fibronectin type III repeat (FN1) of NCAM is required for polyST recognition and the polysialylation of the N-glycans on the adjacent Ig5 domain. In this work, we highlight the importance of an FN1 acidic patch in polyST recognition and also reveal that the polySTs are required to interact with sequences in the Ig5 domain for polysialylation to occur. We find that features of the Ig5 domain of the olfactory cell adhesion molecule (OCAM) are responsible for its lack of polysialylation. Specifically, two basic OCAM Ig5 residues (Lys and Arg) found near asparagines equivalent to those carrying the polysialylated N-glycans in NCAM substantially decrease or eliminate polysialylation when used to replace the smaller and more neutral residues (Ser and Asn) in analogous positions in NCAM Ig5. This decrease in polysialylation does not reflect altered glycosylation but instead is correlated with a decrease in polyST-NCAM binding. In addition, inserting non-conserved OCAM sequences into NCAM Ig5, including an "extra" N-glycosylation site, decreases or completely blocks NCAM polysialylation. Taken together, these results indicate that the polySTs not only recognize an acidic patch in the FN1 domain of NCAM but also must contact sequences in the Ig5 domain for polysialylation of Ig5 N-glycans to occur.

Sequences at the interface of the fifth immunoglobulin domain and first fibronectin type III repeat of the neural cell adhesion molecule are critical for its polysialylation.

Polysialic acid is an anti-adhesive glycan that modifies a select group of mammalian proteins. The primary substrate of the polysialyltransferases (polySTs) is the neural cell adhesion molecule (NCAM). Polysialic acid negatively regulates cell adhesion, is required for proper brain development, and is expressed in specific areas of the adult brain where it promotes on-going cell migration and synaptic plasticity. The first fibronectin type III repeat (FN1) of NCAM is required for polysialylation of the N-glycans on the adjacent immunoglobulin-like domain (Ig5), and acidic residues on the surface of FN1 play a role in polyST recognition. Recent work demonstrated that the FN1 domain from the unpolysialylated olfactory cell adhesion molecule (OCAM) was able to partially replace NCAM FN1 (Foley, D. A., Swartzentruber, K. G., Thompson, M. G., Mendiratta, S. S., and Colley, K. J. (2010) J. Biol. Chem. 285, 35056-35067). Here we demonstrate that individually replacing three identical regions shared by NCAM and OCAM FN1, (500)PSSP(503) (PSSP), (526)GGVPI(530) (GGVPI), and (580)NGKG(583) (NGKG), dramatically reduces NCAM polysialylation. In addition, we show that the polyST, ST8SiaIV/PST, specifically binds NCAM and that this binding requires the FN1 domain. Replacing the FN1 PSSP sequences and the acidic patch residues decreases NCAM-polyST binding, whereas replacing the GGVPI and NGKG sequences has no effect. The location of GGVPI and NGKG in loops that flank the Ig5-FN1 linker and the proximity of PSSP to this linker suggest that GGVPI and NGKG sequences may be critical for stabilizing the Ig5-FN1 linker, whereas PSSP may play a dual role maintaining the Ig5-FN1 interface and a polyST recognition site.

Sequences from the first fibronectin type III repeat of the neural cell adhesion molecule allow O-glycan polysialylation of an adhesion molecule chimera.

Polysialic acid is a developmentally regulated, anti-adhesive polymer that is added to N-glycans on the fifth immunoglobulin domain (Ig5) of the neural cell adhesion molecule (NCAM). We found that the first fibronectin type III repeat (FN1) of NCAM is required for the polysialylation of N-glycans on the adjacent Ig5 domain, and we proposed that the polysialyltransferases recognize specific sequences in FN1 to position themselves for Ig5 N-glycan polysialylation. Other studies identified a novel FN1 acidic surface patch and α-helix that play roles in NCAM polysialylation. Here, we characterize the contribution of two additional FN1 sequences, Pro(510)-Tyr(511)-Ser(512) (PYS) and Gln(516)-Val(517)-Gln(518) (QVQ). Replacing PYS or the acidic patch dramatically decreases the O-glycan polysialylation of a truncated NCAM protein, and replacing the α-helix or QVQ shifts polysialic acid to FN1 O-glycans in full-length NCAM. We also found that the FN1 domain of the olfactory cell adhesion molecule, a homologous but unpolysialylated protein, could partially replace NCAM FN1. Inserting Pro(510)-Tyr(511) eliminated N-glycan polysialylation and enhanced O-glycosylation of an NCAM- olfactory cell adhesion molecule chimera, and inserting other FN1 sequences unique to NCAM, predominantly the acidic patch, created a new polysialyltransferase recognition site. Taken together, our results highlight the role of the FN1 α-helix and QVQ sequences in N-glycan polysialylation and demonstrate that the acidic patch primarily functions in O-glycan polysialylation.

Structure and mutagenesis of neural cell adhesion molecule domains: evidence for flexibility in the placement of polysialic acid attachment sites.

The addition of alpha2,8-polysialic acid to the N-glycans of the neural cell adhesion molecule, NCAM, is critical for brain development and plays roles in synaptic plasticity, learning and memory, neuronal regeneration, and the growth and invasiveness of cancer cells. Our previous work indicates that the polysialylation of two N-glycans located on the fifth immunoglobulin domain (Ig5) of NCAM requires the presence of specific sequences in the adjacent fibronectin type III repeat (FN1). To understand the relationship of these two domains, we have solved the crystal structure of the NCAM Ig5-FN1 tandem. Unexpectedly, the structure reveals that the sites of Ig5 polysialylation are on the opposite face from the FN1 residues previously found to be critical for N-glycan polysialylation, suggesting that the Ig5-FN1 domain relationship may be flexible and/or that there is flexibility in the placement of Ig5 glycosylation sites for polysialylation. To test the latter possibility, new Ig5 glycosylation sites were engineered and their polysialylation tested. We observed some flexibility in glycosylation site location for polysialylation and demonstrate that the lack of polysialylation of a glycan attached to Asn-423 may be in part related to a lack of terminal processing. The data also suggest that, although the polysialyltransferases do not require the Ig5 domain for NCAM recognition, their ability to engage with this domain is necessary for polysialylation to occur on Ig5 N-glycans.

Identification of sequences in the polysialyltransferases ST8Sia II and ST8Sia IV that are required for the protein-specific polysialylation of the neural cell adhesion molecule, NCAM.

The polysialyltransferases ST8Sia II and ST8Sia IV polysialylate the glycans of a small subset of mammalian proteins. Their most abundant substrate is the neural cell adhesion molecule (NCAM). An acidic surface patch and a novel alpha-helix in the first fibronectin type III repeat of NCAM are required for the polysialylation of N-glycans on the adjacent immunoglobulin domain. Inspection of ST8Sia IV sequences revealed two conserved polybasic regions that might interact with the NCAM acidic patch or the growing polysialic acid chain. One is the previously identified polysialyltransferase domain (Nakata, D., Zhang, L., and Troy, F. A. (2006) Glycoconj. J. 23, 423-436). The second is a 35-amino acid polybasic region that contains seven basic residues and is equidistant from the large sialyl motif in both polysialyltransferases. We replaced these basic residues to evaluate their role in enzyme autopolysialylation and NCAM-specific polysialylation. We found that replacement of Arg(276)/Arg(277) or Arg(265) in the polysialyltransferase domain of ST8Sia IV decreased both NCAM polysialylation and autopolysialylation in parallel, suggesting that these residues are important for catalytic activity. In contrast, replacing Arg(82)/Arg(93) in ST8Sia IV with alanine substantially decreased NCAM-specific polysialylation while only partially impacting autopolysialylation, suggesting that these residues may be particularly important for NCAM polysialylation. Two conserved negatively charged residues, Glu(92) and Asp(94), surround Arg(93). Replacement of these residues with alanine largely inactivated ST8Sia IV, whereas reversing these residues enhanced enzyme autopolysialylation but significantly reduced NCAM polysialylation. In sum, we have identified selected amino acids in this conserved polysialyltransferase polybasic region that are critical for the protein-specific polysialylation of NCAM.

Membrane topology of the endoplasmic reticulum to Golgi transport factor Erv29p.

Secretory proteins are transported from the endoplasmic reticulum to the Golgi apparatus via COPII-coated intermediates. Yeast Erv29p is a transmembrane protein cycling between these compartments. It is conserved across species, with one ortholog found in each genome studied, including the surf-4 protein in mammals. Yeast Erv29p acts as a receptor, loading a specific subset of soluble cargo, including glycosylated alpha factor pheromone precursor and carboxypeptidase Y, into vesicles. As the eukaryotic secretory pathway is highly conserved, mammalian surf-4 may perform a similar role in the transport of unknown substrates. Here we report the membrane topology of yeast Erv29p, which we solved by minimally invasive cysteine accessibility scanning using thiol-specific biotinylation and fluorescent labeling methods. Erv29p contains four transmembrane domains with both termini exposed to the cytosol. Two luminal loops may contain a recognition site for hydrophobic export signals on soluble cargo.

Use of a panel of novel genes for differentiating breast cancer from non-breast tissues.

Existing serum markers for breast cancer such as CA 15-3, BR 27.29 and CEA lack sensitivity and specificity. The aim of this study was to evaluate the value of new putative breast-specific markers for differentiating breast cancer from non-breast tissues. Expression of mammaglobin A (MGA), B726P, small breast epithelial mucin (SBEM) and MUC1 was measured by RT-PCR. MGA mRNA was detected in 86/162 (60%) breast cancers but in only 1/32 (3%) non-breast tissues; B726P was detected in 44/108 (41%) breast cancers but in none of 20 non-breast tissues, while SBEM was present in 52/103 (51%) breast cancers but in only 1/26 non-breast cancer tissues. In contrast to these novel markers, the established breast cancer marker MUC1 was detected in 72/99 (73%) breast cancers and in 22/32 (59%) of non-breast tissues. Combining MGA with B726P separated breast cancer from non-breast tissue with a sensitivity of 71% and a specificity of 95% while combining MGA with SBEM differentiated breast cancer from non-breast tissues with a sensitivity of 76% and a specificity of 89%. Genes such as MGA, B726P and SBEM that are expressed relatively exclusively in breast tissue are potential new markers for breast cancer.

Raised plasma endostatin levels correlate inversely with breast cancer angiogenesis.

BACKGROUND: Angiogenesis is essential for solid tumors, such as breast cancer, to grow. The effect of surgical removal of breast tumors on plasma endostatin and vascular endothelial growth factor (VEGF) levels was evaluated. Tumor tissues were analyzed for expression of Intratumoral microvessel density (IMVD) and endostatin. The effect of VEGF and endostatin in inducing apoptosis on human liver microvascular endothelial cells (HLMVEC) was investigated. MATERIALS AND METHODS: Plasma from healthy volunteers, patients with fibroadenomas and breast cancer patients were assayed for endostatin and VEGF via immunoassay, pre-operatively and four weeks post-operatively. Expression of endostatin in tumor tissue was determined by Western blotting. IMVD was assessed following immunohistochemical staining with anti-CD34 antibody. RESULTS: Plasma endostatin levels, in breast cancer patients, were significantly elevated (P = 0.015) in the post-operative (60.59 +/- 7.70 etag/ml) compared with the pre-operative group (30.62 +/- 4.54 etag/ml) and with normal age-matched controls (34.97 +/- 3.76 etag/ml). In patients with high pre-operative plasma endostatin value, IMVD was decreased to 20.1 +/- 3.2 counts compared with 41.9 +/- 5.4 counts in those with low pre-operative endostatin value (P = 0.006). Neither plasma endostatin nor VEGF levels correlated with routine clinico-pathological parameters. Endostatin induced endothelial cell apoptosis and modulated the cytoprotective effect of VEGF in HLMVEC survival. CONCLUSIONS: Plasma endostatin levels are increased in patients following surgical removal of the primary tumor. The decreased IMVD seen in patients with higher endostatin levels may be due to the apoptosis-inducing effect of endostatin on microvascular endothelial cells.