Polyplex-embedding in polyelectrolyte multilayers for gene delivery

In this work, incorporation of plasmid DNA, pre-complexed with PEI, into polyelectrolyte multilayers has been studied to further develop platforms for local gene delivery. Polyplex embedding in synthetic and naturally degradable architectures was efficient for transfection of human hepato-cellular carcinoma cells.     

Sib pair linkage analysis of renin gene haplotypes in human essential hypertension

Summary Although essential arterial hypertension is believed to have a strong genetic predisposition, the gene(s) responsible are unknown. The mechanisms underlying the regulation of blood pressure and experimental studies place the renin gene among the main candidate genes that need to be tested in humans. We tested the hypothesis of a linkage between the renin gene and essential hypertension using the affected sib pair method. Siblings (133 subjects, 52.1±10.9 years) from 57 families were selected for sustained hypertension (160.7 ± 22.9/99.5 ± 12.8 mmHg with 80% of patients under antihypertensive treatment), of early onset (40.7 ± 12.0 years), in the absence of obesity, diabetes mellitus, and secondary hypertension. Eight renin haplotypes were generated from three diallelic renin restriction fragment length polymorphisms (RFLPs) (TaqI, Hinfi, HindIII) located throughout the renin gene. The allelic concordance between the sib pairs was analyzed by identity by state relationships for 98 sib pairs (41 for 41 couples, 39 for 13 trios, 18 for 3 quartets). Allelic frequencies in the 57 hypertensive probands were similar to those observed among 102 hypertensive subjects studied previously. Six of eight possible haplotypes were observed, the informativity of the marker corresponded to 70% of heterozygosity. Allelic concordance for all sib pairs according to sibship size was not significantly different from that expected under the hypothesis of no linkage (t = 0.52, P = 0.15) reflecting only a small excess of renin alleles shared by the hypertensive sibs (1.44 ± 0.6 vs 1.36 ± 0.6). Likewise the linkage hypothesis was unsupported by weighted estimates to correct for possible bias due to large sibship size. Thus, the sib pair analysis suggests that the renin gene does not have a frequent role in the pathogenesis of essential hypertension; further more powerful linkage studies or other approaches will be needed to detect contributions at the renin locus to the heritability of essential hypertension.     

Modulation of the urokinase receptor in human colon cell lines by N,N-dimethylformamide

The present study documents the effect of the planar, polar differentiation promoter N,N-dimethylformamide (DMF) on urokinase binding to colon carcinoma cells. Exposure of the colon carcinoma cell lines to the agent resulted in enhanced specific binding of radioactive urokinase to all cells tested. Insulin binding to the cells was, however, unaffected by DMF. A DMF exposure period of 45 h was required to observe maximum urokinase binding to two representative cell lines FET and RKO. Optimal stimulation of both cell lines occurred with 0.8% DMF. Scatchard analysis revealed the dissociation constants to be unchanged by the agent with the increased binding of radioactive plasminogen activator reflecting an up-regulation of binding sites. In this regard, the cell line RKO upon exposure to DMF, displayed approx. 700,000 receptors/cell, the highest value published, to date, for any cell line.     

Inositol tetrakisphosphate isomers and elevation of cytosolic Ca2+ in vasopressin-stimulated insulin-secreting RINm5F cells.

Signal generation during the stimulation of insulin secretion by arginine vasopressin (AVP) was investigated in RINm5F cells. AVP (0.1 microM) caused a biphasic cytosolic Ca2+ ([Ca2+]i) rise, namely a rapid transient marked elevation after stimulation followed by a series of oscillations. In the absence of extracellular Ca2+, the sustained oscillations were abolished, while the initial [Ca2+]i transient was only partly decreased, indicating that the former are due to Ca2+ influx and the latter due mainly to mobilization from internal Ca2+ stores. AVP also evoked a transient depolarization of the average membrane potential. AVP-induced Ca2+ influx during the sustained phase, which was strictly dependent on receptor occupancy, was attenuated by membrane hyperpolarization with diazoxide. However, blockade of Ca2+ channels of the L- or T-type was ineffective. AVP stimulated production of diacylglycerol and inositol phosphates; for the latter both [3H] inositol labeling and mass determinations were performed. A transient increase in Ins(1,4,5)P3 was followed by a marked enhancement of Ins(1,3,4,5)P4 (8-fold) peaking at 15 s and gradually returning to basal values. Ins(1,3,4,6)P4 and Ins(3,4,5,6)P4 exhibited the most long-lasting augmentation (4- and 1.7-fold, respectively), and therefore correlated best with the period of sustained [Ca2+]i oscillations. InsP5 and InsP6 were not elevated. The effects of AVP, including the stimulation of insulin secretion from perifused cells, were obliterated by a V1 receptor antagonist. In conclusion, AVP induces protracted [Ca2+]i elevation in RINm5F cells which is associated with long-lasting increases in InsP4 isomers. The accumulation of InsP4 isomers reflects receptor occupancy and accelerated metabolism of the inositol phosphates. Activation of second messenger-operated Ca2+ channels is not necessarily implicated because of the attenuating effect of membrane hyperpolarization.     

Unbiased proteomic profiling of host cell extracellular vesicle composition and dynamics upon HIV‐1 infection

Cells release diverse types of extracellular vesicles (EVs), which transfer complex signals to surrounding cells. Specific markers to distinguish different EVs (e.g. exosomes, ectosomes, enveloped viruses like HIV) are still lacking. We have developed a proteomic profiling approach for characterizing EV subtype composition and applied it to human Jurkat T cells. We generated an interactive database to define groups of proteins with similar profiles, suggesting release in similar EVs. Biochemical validation confirmed the presence of preferred partners of commonly used exosome markers in EVs: CD81/ADAM10/ITGB1, and CD63/syntenin. We then compared EVs from control and HIV‐1‐infected cells. HIV infection altered EV profiles of several cellular proteins, including MOV10 and SPN, which became incorporated into HIV virions, and SERINC3, which was re‐routed to non‐viral EVs in a Nef‐dependent manner. Furthermore, we found that SERINC3 controls the surface composition of EVs. Our workflow provides an unbiased approach for identifying candidate markers and potential regulators of EV subtypes. It can be widely applied to in vitro experimental systems for investigating physiological or pathological modifications of EV release.     

Fiber background rejection and crystal over-response compensation for GaN based in vivo dosimetry

For dosimetric measurements using an implantable optical fiber probe with GaN (Gallium Nitride) scintillator as radioluminescence (RL) transducer, a bi-channel method is proposed to reject the background contribution of the irradiated fiber segment. It is based on spectral differences between the narrow-band light emission from GaN and the large-band background from the irradiated optical fiber. Experimental validation of this method using 6 MV photon beam has shown that the remaining background contribution after subtraction is below 1.2% for square field sizes ranging from 3 cm to 20 cm. Furthermore, a compensation method for the over-response of GaN is also proposed, since GaN is not tissue equivalent. The over-response factor of GaN exhibits a linear increase with square field aperture and depends on depth from phantom surface. This behaviour is modelled to allow compensation in specific conditions. The proposed method has been evaluated and has shown a maximum deviation of 3% for a 6 MV photon beam and 1% for an 18 MV photon beam at a depth beyond the build-up region.     

Syndromic Algorithms for Detection of Gambiense Human African Trypanosomiasis in South Sudan

Background

Active screening by mobile teams is considered the best method for detecting human African trypanosomiasis (HAT) caused by Trypanosoma brucei gambiense but the current funding context in many post-conflict countries limits this approach. As an alternative, non-specialist health care workers (HCWs) in peripheral health facilities could be trained to identify potential cases who need testing based on their symptoms. We explored the predictive value of syndromic referral algorithms to identify symptomatic cases of HAT among a treatment-seeking population in Nimule, South Sudan.

Methodology/Principal Findings

Symptom data from 462 patients (27 cases) presenting for a HAT test via passive screening over a 7 month period were collected to construct and evaluate over 14,000 four item syndromic algorithms considered simple enough to be used by peripheral HCWs. For comparison, algorithms developed in other settings were also tested on our data, and a panel of expert HAT clinicians were asked to make referral decisions based on the symptom dataset. The best performing algorithms consisted of three core symptoms (sleep problems, neurological problems and weight loss), with or without a history of oedema, cervical adenopathy or proximity to livestock. They had a sensitivity of 88.9–92.6%, a negative predictive value of up to 98.8% and a positive predictive value in this context of 8.4–8.7%. In terms of sensitivity, these out-performed more complex algorithms identified in other studies, as well as the expert panel. The best-performing algorithm is predicted to identify about 9/10 treatment-seeking HAT cases, though only 1/10 patients referred would test positive.

Conclusions/Significance

In the absence of regular active screening, improving referrals of HAT patients through other means is essential. Systematic use of syndromic algorithms by peripheral HCWs has the potential to increase case detection and would increase their participation in HAT programmes. The algorithms proposed here, though promising, should be validated elsewhere.     

Systemic Down-Regulation of Delta-9 Desaturase Promotes Muscle Oxidative Metabolism and Accelerates Muscle Function Recovery following Nerve Injury

The progressive deterioration of the neuromuscular axis is typically observed in degenerative conditions of the lower motor neurons, such as amyotrophic lateral sclerosis (ALS). Neurodegeneration in this disease is associated with systemic metabolic perturbations, including hypermetabolism and dyslipidemia. Our previous gene profiling studies on ALS muscle revealed down-regulation of delta-9 desaturase, or SCD1, which is the rate-limiting enzyme in the synthesis of monounsaturated fatty acids. Interestingly, knocking out SCD1 gene is known to induce hypermetabolism and stimulate fatty acid beta-oxidation. Here we investigated whether SCD1 deficiency can affect muscle function and its restoration in response to injury. The genetic ablation of SCD1 was not detrimental per se to muscle function. On the contrary, muscles in SCD1 knockout mice shifted toward a more oxidative metabolism, and enhanced the expression of synaptic genes. Repressing SCD1 expression or reducing SCD-dependent enzymatic activity accelerated the recovery of muscle function after inducing sciatic nerve crush. Overall, these findings provide evidence for a new role of SCD1 in modulating the restorative potential of skeletal muscles.     

Microarray Analysis of Microbiota of Gingival Lesions in Noma Patients

Noma (cancrum oris) is a gangrenous disease of unknown etiology affecting the maxillo-facial region of young children in extremely limited resource countries. In an attempt to better understand the microbiological events occurring during this disease, we used phylogenetic and low-density microarrays targeting the 16S rRNA gene to characterize the gingival flora of acute noma and acute necrotizing gingivitis (ANG) lesions, and compared them to healthy control subjects of the same geographical and social background. Our observations raise doubts about Fusobacterium necrophorum, a previously suspected causative agent of noma, as this species was not associated with noma lesions. Various oral pathogens were more abundant in noma lesions, notably Atopobium spp., Prevotella intermedia, Peptostreptococcus spp., Streptococcus pyogenes and Streptococcus anginosus. On the other hand, pathogens associated with periodontal diseases such as Aggregatibacter actinomycetemcomitans, Capnocytophaga spp., Porphyromonas spp. and Fusobacteriales were more abundant in healthy controls. Importantly, the overall loss of bacterial diversity observed in noma samples as well as its homology to that of ANG microbiota supports the hypothesis that ANG might be the immediate step preceding noma.