Functional and physical interaction between the mismatch repair and FA-BRCA pathways

Fanconi anemia (FA) is a rare genetic disorder characterized by bone marrow failure and an increased risk for leukemia and cancer. Fifteen proteins thought to function in the repair of DNA interstrand crosslinks (ICLs) comprise what is known as the FA-BRCA pathway. Activation of this pathway leads to the monoubiquitylation and chromatin localization of FANCD2 and FANCI. It has previously been shown that FANCJ interacts with the mismatch repair (MMR) complex MutLα. Here we show that FANCD2 interacts with the MMR proteins MSH2 and MLH1. FANCD2 monoubiquitylation, foci formation and chromatin loading are greatly diminished in MSH2-deficient cells. Human or mouse cells lacking MSH2 or MLH1 display increased sensitivity and radial formation in response to treatment with DNA crosslinking agents. Studies in human cell lines and Drosophila mutants suggest an epistatic relationship between FANCD2, MSH2 and MLH1 with regard to ICL repair. Surprisingly, the interaction between MSH2 and MLH1 is compromised in multiple FA cell lines, and FA cell lines exhibit deficient MMR. These results suggest a significant role for MMR proteins in the activation of the FA pathway and repair of ICLs. In addition, we provide the first evidence for a defect in MMR in FA cell lines.     

Antibacterial activity of a synthetic peptide that mimics the LPS binding domain of Indian mud crab, Scylla serrata anti-lipopolysaccharide factor (SsALF) also involved in the modulation of vaginal immune functions through NF-kB signaling

Recently the cDNA coding for anti-lipopolysaccharide factor (ALF) has been identified from the Indian mud crab, Scylla serrata and has been named S. serrata anti-lipopolysaccharide factor (SsALF). SsALF protein sequence demonstrated the presence of two highly conserved cystine residues between which the putative lipopolysaccharide (LPS) binding domain is known to be located. In this study, we have designed and synthesized a 24 amino acid linear (lSsALF24) and a cyclic (cSsALF24) peptides based on this putative LPS binding domain and demonstrated the ability of these peptides to bind to LPS. The peptides were active against vaginal pathogens demonstrated by MIC, CFU and phagocytosis assays. cSsALF24 did not show toxicity to human vaginal epithelial cells (HeLa-S3), macrophages and rabbit erythrocytes even at high concentration (64.64 μM). Flow cytometry results demonstrated that cSsALF24 peptide suppressed LPS induced phagocytosis of FITC labeled E. coli. HeLa cells were stimulated with LPS (10 μg/ml) alone for 6 h or after two washings with PBS, treated for 1 h with cSsALF24 (64.64 μM). After washing, the cells were cultured for 24 h in fresh media. The spent media as well as cells were collected for the determination of cytokine/chemokine levels such as interleukin-6 (IL-6) interleukin-8 (IL-8), monocyte chemotactic protein-1 (MCP-1) and interleukin-1α (IL-1α) using ELISA and RT-PCR respectively. Similar results were obtained with LPS stimulated cells treated with c/nSsALF24 or unstimulated cells treated with c/nSsALF24. The expression of cytokine/chemokines and mRNA’s coding these proteins were unaffected in c/nSsALF24 treated cells. In contrast, in LPS stimulated cells, the expression levels of these molecules were up-regulated via the induction of nuclear factor kappa-B (NF-kB) levels. However, the expression of these pro-inflammatory markers was decreased in LPS stimulated cells following the treatment with cSsALF24, attributing anti-inflammatory potential of the peptide. Collectively, these findings suggest that cSsALF24 might regulate the vaginal epithelial cell immune responses indirectly through modulation of LPS-TLR4 binding in NF-kB pathway.     

Effects of citalopram on serotonin and CRF systems in the midbrain of primates with differences in stress sensitivity

This chapter reviews the neurobiological effects of stress sensitivity and s-citalpram (CIT) treatment observed in our nonhuman primate model of functional hypothalamic amenorrhea (FHA). This type of infertility, also known as stress-induced amenorrhea, is exhibited by cynomolgus macaques. In small populations, some individuals are stress-sensitive (SS) and others are highly stress-resilient (HSR). The SS macaques have suboptimal secretion of estrogen and progesterone during normal menstrual cycles. SS monkeys also have decreased serotonin gene expression and increased CRF expression compared to HSR monkeys. Recently, we found that CIT treatment improved ovarian steroid secretion in SS monkeys, but had no effect in HSR monkeys. Examination of the serotonin system revealed that SS monkeys had significantly lower Fev (fifth Ewing variant, rodent Pet1), TPH2 (tryptophan hydroxylase 2), 5HT1A autoreceptor and SERT (serotonin reuptake transporter) expression in the dorsal raphe than SR monkeys. However, CIT did not alter the expression of either Fev, TPH2, SERT or 5HT1A mRNAs. In contrast, SS monkeys tended to have a higher density of CRF fiber innervation of the dorsal raphe than HSR monkeys, and CIT significantly decreased the CRF fiber density in SS animals. In addition, CIT increased CRF-R2 gene expression in the dorsal raphe. We speculate that in a 15-week time frame, the therapeutic effect of S-citalopram may be achieved through a mechanism involving extracellular serotonin inhibition of CRF and stimulation of CRF-R2, rather than alteration of serotonin-related gene expression.     

Novel wheat protein films as substrates for tissue engineering

This paper demonstrates that gliadin-free wheat glutenin can be an excellent biomaterial for tissue-engineering applications, better than poly(lactic acid) (PLA). Although plant proteins are more readily available than collagen and silk, limited studies have been conducted on understanding the potential of using plant proteins as biomaterials. Wheat proteins have not been used for tissue engineering due to the cytotoxicity of gliadin. In this research, wheat gluten, glutenin and gliadin were used to develop films and the mechanical properties, water stability and ability of the films to promote the attachment, growth and viability of osteoblast cells was studied in comparison to PLA films. The wheat protein films have good strength ranging from 8 to 30 MPa. Gliadin films experience about 50% weight loss whereas glutenin films have about 90% weight loss after being in water (pH 7.2) for 15 days at 37°C. Gliadin is cytotoxic and the presence of gliadin restricts the cell proliferation on wheat gluten films. However, gliadin-free glutenin films show a higher rate of proliferation of osteoblast cells than the PLA films. Wheat gluten promises to be a potential substrate for tissue engineering and other medical applications.     

Role of 14-bp insertion/deletion polymorphism in HLA-G among Indian women with recurrent spontaneous abortions

Human leukocyte antigen (HLA)-G is predominantly expressed on the extravillous cytotrophoblasts at the fetal-maternal interface. The 14-bp polymorphism in exon 8 is associated with HLA-G messenger ribonucleic acid (mRNA) stability and isoform alternative splicing patterns, thereby influencing the functionality of HLA-G in pregnancy. We analysed the 14-bp indel polymorphism in 143 recurrent spontaneous abortions (RSAs) and 150 control couples. We did not find any significant difference in the 14-bp insertion/deletion allele frequencies among the RSA and control couples. Analysis for increased sharing of the polymorphism in the RSA and the control couples also did not show any significant difference. However, we found an increase in the frequency of the 14-bp deletion homozygotes in the RSA women, which could lead to extremely high levels of soluble HLA-G (sHLA-G).     

Detection of cardiac myosin heavy chain-α-specific CD4 cells by using MHC class II/IA(k) tetramers in A/J mice

A/J mice bearing the H-2 allele IA(k) are highly susceptible to autoimmune myocarditis induced with cardiac myosin heavy chain (Myhc)-α 334-352, whereas B10.A mice carrying a similar allele IA(k) are relatively resistant, suggesting that the generation of Myhc-α-reactive T cell repertoires is influenced by genetic background. To enumerate the precursor frequencies of Myhc-α-specific CD4 T cells, we sought to create IA(k) tetramers for Myhc-α 334-352. Tetramers were created using approaches that involve covalent tethering of individual peptide sequences or exogenous loading of peptides into empty IA(k) molecules by peptide-exchange reaction. Using ribonuclease 43-56 tetramers as controls, we demonstrated that by flow cytometry (FC), Myhc-α 334-352 tetramers specifically bind myosin-reactive T cells. CD4 cells isolated from A/J mice immunized with Myhc-α 334-352 were used to optimize conditions for tetramer staining, and neuraminidase treatment prior to tetramer staining permitted the detection of Myhc-α-specific cells ex vivo. The reagents are useful tools for monitoring the frequency of Myhc-α-reactive CD4 cells and to determine their pathogenic potential at a single cell level by FC.     

Evaluation of tracheal tube introducers in simulated difficult intubation*

In a randomised cross‐over study, 72 anaesthetists attempted to place Pro‐Breathe, new Portex, and Frova single‐use tracheal tube introducers and an Eschmann multiple‐use introducer in the trachea of a manikin set to simulate a grade 3 laryngeal view. Successful placement (proportion, 95% confidence interval) of either the Frova (78%, 67–86%) or the Eschmann introducer (64%, 52–74%) was significantly more likely (p < 0.0001) than with the Pro‐Breathe (4%, 1–12%) or the new Portex introducer (13%, 7–22%). The difference between the success rates for the Frova and the Eschmann introducers (p = 0.08) was not significant. A separate experiment revealed that the peak force that could be exerted by the Pro‐Breathe, new Portex and Frova single‐use introducers were three to six times greater than that which could be exerted by the Eschmann introducer (p < 0.0001). The single‐use introducers are more likely to cause tissue trauma during placement, particularly if held close to the tip.     

Outcomes of Simultaneous Kidney-Pancreas Transplantation With Positive Cross-Match

We studied 72 consecutive simultaneous pancreas kidney transplant (SPKT) recipients. There were 14 patients with positive pretransplant cross-matches (positive CDC- B cell and/or positive flow T or B cross-match). The control group included all 58 SPKT recipients with a negative crossmatch. The study group received induction with low dose intravenous immunoglobulin (IVIg), rabbit antithymocyte globulin (rATG; total dose 6 mg/kg), or alemtuzumab (30 mg single dose) and maintenance with tacrolimus, mycophenolate mofetil (MMF), and corticosteroids. The control group was treated similarly, but with steroid avoidance and no IVIg. Biopsy-proven acute rejection (BPAR) of the kidney allograft occurred in 7 study patients (50%) compared with 10% in the control group (P = .022). One patient experienced acute cellular rejection (ACR); the other 6 (43%), antibody-mediated rejection (AMR). None of the cross-match negative patients had AMR (P = .001). The mean follow-up period was 18.7 months in the study group, and 18.3 months in the control group. The 1-year actuarial patient survival was 91.7% in the study group and 97% in the control group. Kidney allograft survival was 91.7% in the study group and 95.2% in the control group. Pancreas allograft survival was 76.9% in study group and 89.6% in the control group (P = .088). We concluded that patients with a positive pretransplant CDC-B cross-match and/or positive flow cross-match have an increased risk of AMR; more intensive desensitization is needed with low-dose IVIg and induction with either rATG or alemtuzumab.     

Challenges to effective crisis management: using information and communication technologies to coordinate emergency medical services and emergency department teams

OBJECTIVE: The purpose of this study is to identify the major challenges to coordination between emergency department (ED) teams and emergency medical services (EMS) teams. DESIGN: We conducted a series of focus groups involving both ED and EMS team members using a crisis scenario as the basis of the focus group discussion. We also collected organizational workflow data. RESULTS: We identified three major challenges to coordination between ED and EMS teams including ineffectiveness of current information and communication technologies, lack of common ground, and breakdowns in information flow. DISCUSSION: The three challenges highlight the importance of designing systems from socio-technical perspective. In particular, these inter-team coordination systems must support socio-technical issues such as awareness, context, and workflow between the two teams.