Identifying Pathways for Improving Household Food Self-Sufficiency Outcomes in the Hills of Nepal

Maintaining and improving household food self-sufficiency (FSS) in mountain regions is an ongoing challenge. There are many facets to the issue, including comparatively high levels of land fragmentation, challenging terrain and transportation bottlenecks, declining labor availability due to out-migration, and low technical knowledge, among others. Using a nonparametric multivariate approach, we quantified primary associations underlying current levels of FSS in the mid-hills of Nepal. A needs assessment survey was administered to 77 households in Lungaun (Baglung District), Pang (Parbat District), and Pathlekhet (Myagdi District), with a total of 80 variables covering five performance areas; resulting data were analyzed using Classification and Regression Trees. The most parsimonious statistical model for household FSS highlighted associations with agronomic management, including yields of maize and fingermillet within a relay cropping system and adoption of improved crop cultivars. Secondary analyses of the variables retained in the first model again focused primarily on crop and livestock management. It thus appears that continued emphasis on technical agricultural improvements is warranted, independent of factors such as land holding size that, in any case, are very difficult to change through development interventions. Initiatives to increase household FSS in the mid-hills of Nepal will benefit from placing a primary focus on methods of agricultural intensification to improve crop yields and effective technology transfer to increase adoption of these methods.     

CD47 does not mediate amyloid-β(1–42) protofibril-stimulated microglial cytokine release

Neuroinflammation triggered by accumulation of amyloid-β protein (Aβ) is a significant component of the Alzheimer’s disease (AD) brain. Senile plaques composed of Aβ attract and activate microglia cells resulting in cytokine secretion and a proinflammatory environment. The mechanism by which Aβ activates microglia is complex and involves numerous cellular components. One receptor potentially involved in Aβ recognition and the ensuing microglia proinflammatory response is CD47. Since there is significant interest in soluble aggregated Aβ species, we sought to determine if CD47 plays a key role in microglia cytokine release stimulated by soluble Aβ(1–42) protofibrils. Pretreatment of primary murine microglia with the CD47 antagonist peptide 4N1K significantly and potently inhibited both tumor necrosis factor-α (TNFα) and interleukin-1β (IL-1β) secretion stimulated by Aβ(1–42) protofibrils. 4N1K displayed toxicity to the microglia but only at concentrations much higher than the observed inhibition. Surprisingly, 4N1K also potently inhibited TNFα secretion triggered by lipopolysaccharide which is not known to signal through CD47. Treatment of the microglia with a neutralizing anti-CD47 antibody failed to block the Aβ protofibril response even though comparable samples were completely inhibited by 4N1K. Finally, Aβ(1–42) protofibrils stimulated similar levels of secreted TNFα production in both wild-type and CD47^−/− microglia and 4N1K still potently inhibited the Aβ protofibril response even in the CD47^−/− microglia. The overall findings demonstrated that the microglial proinflammatory response to Aβ(1–42) protofibril is not dependent on CD47 and that 4N1K exhibits CD47-independent inhibitory activity.     

Application of azide-alkyne cycloaddition 'click chemistry' for the synthesis of Grb2 SH2 domain-binding macrocycles

Copper (I) promoted [3+2] Huisgen cycloaddition of azides with terminal alkynes was used to prepare triazole-containing macrocycles based on the Grb2 SH2 domain-binding motif, 'Pmp-Ac(6)c-Asn', where Pmp and Ac(6)c stand for 4-phosphonomethylphenylalanine and 1-aminocyclohexanecarboxylic acid, respectively. When cycloaddition reactions were conducted at 1mM substrate concentrations, cyclization of monomeric units occurred. At 2mM substrate concentrations the predominant products were macrocyclic dimers. In Grb2 SH2 domain-binding assays the monomeric (S)-Pmp-containing macrocycle exhibited a K(d) value of 0.23microM, while the corresponding dimeric macrocycle was found to have greater than 50-fold higher affinity. The open-chain dimer was also found to have affinity equal to the dimeric macrocycle. This work represents the first application of 'click chemistry' to the synthesis of SH2 domain-binding inhibitors and indicates its potential utility.     

Mechanism of beta-purothionin antimicrobial peptide inhibition by metal ions: molecular dynamics simulation study

Wheat beta-purothionin is a highly potent antimicrobial peptide which, however, is inactivated by metal ions. The key structural properties and mechanisms of inhibition of beta-purothionin were investigated for the first time using unconstrained molecular dynamics simulations in explicit water. A series of simulations were performed to determine effects of temperature and the metal ions. Analyses of the unconstrained simulations allowed the experimentally unavailable structural and dynamic details to be unambiguously examined. The global fold and the alpha1 helix of beta-purothionin are thermally stable and not affected by metal ions. In contrast, the alpha2 helix unfolds with shift of temperature from 300 K and in the presence of metal ions. The network of conserved residues including Arg30 and Lys5 is sensitive to environmental changes and triggers unfolding. Loop regions display high flexibility and elevated dynamics, but are affected by metal ions. Our study provides insights into the mechanism of metal ion-based inhibition.     

A survey of enteric bacteria and protozoans in fresh bovine faeces on New Zealand dairy farms

Aims: To determine the counts and/or prevalence in fresh bovine faeces of Escherichia coli, enterococci, Campylobacter, Salmonella, shiga toxin‐producing E. coli (STEC), Giardia and Cryptosporidium, as inputs to numerical models designed to estimate microbial loadings on pasture grazed by cattle in New Zealand. Methods and Results: In each season over one year, samples of freshly deposited bovine faeces were collected from four New Zealand dairy farms (n = 155), and enumerated for E. coli, enterococci, Campylobacter, Giardia and Cryptosporidium. They were also tested for the presence of Salmonella and STEC. The overall median bacterial counts (g^?1 wet weight) were E. coli– 5·9 × 10⁶; enterococci – 1·3 × 10⁴; Campylobacter– 3·9 × 10⁵. All counts were highly variable within and between samplings, and few seasonal or regional patterns emerged. However, mean Campylobacter counts were consistently higher in spring. No Salmonella spp. was detected, and only two samples were positive for STEC. Cryptosporidium and Giardia were isolated from 5·2% and 4·5% of the samples, respectively, yielding low numbers of (oo)cysts (1–25 g^?1 and 1–17 g^?1, respectively). Conclusions: Fresh bovine faeces are a significant source of E. coli, enterococci and Campylobacter on New Zealand pastures, although numbers are likely to vary markedly between faecal samples. Significance and Impact of the Study: The study provides the first significant set of indicator and pathogen counts for one of the largest sources of faecal contamination of natural waters in New Zealand, and will be used to model these inputs.     

Kaposi’s Sarcoma-Associated Herpesvirus K3 and K5 Proteins Down Regulate Both DC-SIGN and DC-SIGNR

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiological agent of multicentric Castleman’s disease, primary effusion lymphoma and Kaposi’s sarcoma. In this study, we show that like the C-type lectin DC-SIGN, the closely related DC-SIGNR can also enhance KSHV infection. Following infection, they are both targeted for down modulation and our data indicate that the KSHV MARCH-family ubiquitin ligase K5 is mediating this regulation and subsequent targeting for degradation of DC-SIGN and DC-SIGNR in the context of the virus. The closely related viral K3 protein, is also able to target these lectins in exogenous expressions studies, but only weakly during viral infection. In addition to requiring a functional RING-CH domain, several protein trafficking motifs in the C-terminal region of both K3 and K5 are important in regulation of DC-SIGN and DC-SIGNR. Further exploration of this modulation revealed that DC-SIGN is endocytosed from the cell surface in THP-1 monocytes, but degraded from an internal location with minimal endocytosis in HEK-293 cells. Pull-down data indicate that both K3 and K5 preferentially associate with immature forms of the lectins, mediating their ubiquitylation and degradation. Together, these data emphasize the molecular complexities of K3 and K5, while expanding the repertoire of targets of these two viral proteins.     

CCR5 Haplotypes Influence HCV Serostatus in Caucasian Intravenous Drug Users

Background

Up to 90% HIV-1 positive intravenous drug users (IDUs) are co-infected with HCV. Although best recognized for its function as a major co-receptor for cell entry of HIV, CC chemokine receptor 5 (CCR5) has also been implicated in the pathogenesis of HCV infection. Here, we investigated whether CCR5 haplotypes influence HIV-1 and HCV seropositivity among 373 Caucasian IDUs from Estonia.

Methods

Of these IDUs, 56% and 44% were HIV and HCV seropositive, respectively, and 47% were coinfected. 500 blood donors seronegative for HIV and HCV were also evaluated. CCR5 haplotypes (HHA to HHG*2) were derived after genotyping nine CCR2–CCR5 polymorphisms. The association between CCR5 haplotypes with HIV and/or HCV seropositivity was determined using logistic regression analysis. Co-variates included in the models were length of intravenous drug use, HBV serostatus and copy number of CCL3L1, the gene encoding the most potent HIV-suppressive chemokine and ligand for CCR5.

Results

Compared to IDUs seronegative for both HCV and HIV (HCV−/HIV-), IDUs who were HCV+/HIV- and HCV+/HIV+were 92% and 82%, respectively, less likely to possess the CCR5-HHG*1 haplotype, after controlling for co-variates (P_adjusted = 1.89×10⁻⁴ and 0.003, respectively). This association was mostly due to subjects bearing the CCR5 HHE and HHG*1 haplotype pairs. Approximately 25% and<10% of HCV−/HIV- IDUs and HCV−/HIV- blood donors, respectively, possessed the HHE/HHG*1 genotype.

Conclusions

Our findings suggest that HHG*1-bearing CCR5 genotypes influence HCV seropositivity in a group of Caucasian IDUs.     

B-Raf associates with and activates the NHE1 isoform of the Na+/H+ exchanger

The serine/threonine kinase B-Raf is the second most frequently occurring human oncogene after Ras. Mutations of B-Raf occur with the highest incidences in melanoma, and the most common mutant, V600E, renders B-Raf constitutively active. The sodium proton exchanger isoform 1 (NHE1) is a ubiquitously expressed plasma membrane protein responsible for regulating intracellular pH, cell volume, cell migration, and proliferation. A screen of protein kinases that bind to NHE1 revealed that B-Raf bound to the cytosolic regulatory tail of NHE1. Immunoprecipitation of NHE1 from HeLa and HEK cells confirmed the association of B-Raf with NHE1 in vivo. The expressed and purified C-terminal 182 amino acids of the NHE1 protein were also shown to associate with B-Raf protein in vitro. Because treatment with the kinase inhibitor sorafenib decreased NHE1 activity in HeLa and HEK cells, we examined the role of B-Raf in regulating NHE1 in malignant melanoma cells. Melanoma cells with the B-Raf(V600E) mutation demonstrated increased resting intracellular pH that was dependent on elevated NHE1 activity. NHE1 activity after an acute acid load was also elevated in these cell lines. Moreover, inhibition of B-Raf activity by either sorafenib, PLX4720, or siRNA reduction of B-Raf levels abolished ERK phosphorylation and decreased NHE1 activity. These results demonstrate that B-Raf associates with and stimulates NHE1 activity and that B-Raf(V600E) also increases NHE1 activity that raises intracellular pH.