Following Mitochondrial Footprints through a Long Mucosal Path to Lung Cancer

Background

Mitochondrial DNA (mtDNA) mutations are reported in different tumors. However, there is no information on the temporal development of the mtDNA mutations/content alteration and their extent in normal and abnormal mucosa continuously exposed to tobacco smoke in lung cancer patients.

Methodology

We examined the pattern of mtDNA alteration (mtDNA mutation and content index) in 25 airway mucosal biopsies, corresponding tumors and normal lymph nodes obtained from three patients with primary lung cancers. In addition, we examined the pattern of mtDNA mutation in corresponding tumors and normal lymph nodes obtained from eight other patients with primary lung cancers. The entire 16.5 kb mitochondrial genome was sequenced on Affymetrix Mitochip v2.0 sequencing platform in every sample. To examine mtDNA content index, we performed real-time PCR analysis.

Principal Findings

The airway mucosal biopsies obtained from three lung cancer patients were histopathologically negative but exhibited multiple clonal mtDNA mutations detectable in the corresponding tumors. One of the patients was operated twice for the removal of tumor from the right upper and left lower lobe respectively within a span of two years. Both of these tumors exhibited twenty identical mtDNA mutations. MtDNA content increased significantly (P<0.001) in the lung cancer and all the histologically negative mucosal biopsies except one compared to the control lymph node.

Conclusions/Significance:

Our results document the extent of massive clonal patches that develop in lifetime smokers and ultimately give rise to clinically significant cancers. These observations shed light on the extent of disease in the airway of smokers traceable through mtDNA mutation. MtDNA mutation could be a reliable tool for molecular assessment of respiratory epithelium exposed to continuous smoke as well as disease detection and monitoring. Functional analysis of the pathogenic mtDNA mutations may be useful to understand their role in lung tumorigenesis.     

Protein-protein cross-linking and human health: the challenge of elucidating with mass spectrometry

Introduction: In several biomedical research fields, the cross-linking of peptides and proteins has an important impact on health and wellbeing. It is therefore of crucial importance to study this class of post-translational modifications in detail. The huge potential of mass spectrometric technologies in the mapping of these protein-protein cross-links is however overshadowed by the challenges that the field has to overcome.

Areas covered: In this review, we summarize the different pitfalls and challenges that the protein-protein cross-linking field is confronted with when using mass spectrometry approaches. We additionally focus on native disulfide bridges as an example and provide some examples of cross-links that are important in the biomedical field.

Expert commentary: The current flow of methodological improvements, mainly from the chemical cross-linking field, has delivered a significant contribution to deciphering native and insult-induced cross-links. Although an automated data analysis of proteome-wide peptide cross-linking is currently only possible in chemical cross-linking experiments, the field is well on the way towards a more automated analysis of native and insult-induced cross-links in raw mass spectrometry data that will boost its potential in biomedical applications.     

Complexin synchronizes primed vesicle exocytosis and regulates fusion pore dynamics

ComplexinII (CpxII) and SynaptotagminI (SytI) have been implicated in regulating the function of SNARE proteins in exocytosis, but their precise mode of action and potential interplay have remained unknown. In this paper, we show that CpxII increases Ca²⁺-triggered vesicle exocytosis and accelerates its secretory rates, providing two independent, but synergistic, functions to enhance synchronous secretion. Specifically, we demonstrate that the C-terminal domain of CpxII increases the pool of primed vesicles by hindering premature exocytosis at submicromolar Ca²⁺ concentrations, whereas the N-terminal domain shortens the secretory delay and accelerates the kinetics of Ca²⁺-triggered exocytosis by increasing the Ca²⁺ affinity of synchronous secretion. With its C terminus, CpxII attenuates fluctuations of the early fusion pore and slows its expansion but is functionally antagonized by SytI, enabling rapid transmitter discharge from single vesicles. Thus, our results illustrate how key features of CpxII, SytI, and their interplay transform the constitutively active SNARE-mediated fusion mechanism into a highly synchronized, Ca²⁺-triggered release apparatus.     

Trends in the Epidemiology of Pandemic and Non-pandemic Strains of Vibrio parahaemolyticus Isolated from Diarrheal Patients in Kolkata,India

A total of 178 strains of V. parahaemolyticus isolated from 13,607 acute diarrheal patients admitted in the Infectious Diseases Hospital, Kolkata has been examined for serovar prevalence, antimicrobial susceptibility and genetic traits with reference to virulence, and clonal lineages. Clinical symptoms and stool characteristics of V. parahaemolyticus infected patients were analyzed for their specific traits. The frequency of pandemic strains was 68%, as confirmed by group-specific PCR (GS-PCR). However, the prevalence of non-pandemic strains was comparatively low (32%). Serovars O3:K6 (19.7%), O1:K25 (18.5%), O1:KUT (11.2%) were more commonly found and other serovars such as O3:KUT (6.7%), O4:K8 (6.7%), and O2:K3 (4.5%) were newly detected in this region. The virulence gene tdh was most frequently detected in GS-PCR positive strains. There was no association between strain features and stool characteristics or clinical outcomes with reference to serovar, pandemic/non-pandemic or virulence profiles. Ampicillin and streptomycin resistance was constant throughout the study period and the MIC of ampicillin among selected strains ranged from 24 to >256 µg/ml. Susceptibility of these strains to ampicillin increased several fold in the presence of carbonyl cyanide-m-chlorophenyldrazone. The newly reported ESBL encoding gene from VPA0477 was found in all the strains, including the susceptible ones for ampicillin. However, none of the strains exhibited the β-lactamase as a phenotypic marker. In the analysis of pulsed-field gel electrophoresis (PFGE), the pandemic strains formed two different clades, with one containing the newly emerged pandemic strains in this region.     

Decreased sensory responses in osteocalcin null mutant mice imply neuropeptide function

Osteocalcin, the most abundant member of the family of extracellular mineral binding gamma-carboxyglutamic acid proteins is synthesized primarily by osteoblasts. Its affinity for calcium ions is believed to limit bone mineralization. Several of the numerous hormones that regulate synthesis of osteocalcin, including glucocorticoids and parathyroid hormone, are also affected by stressful stimuli that require energy for an appropriate response. Based on our observations of OC responding to stressful sensory stimuli, the expression of OC in mouse and rat sensory ganglia was confirmed. It was thus hypothesized that the behavioral responses of the OC knockout mouse to stressful sensory stimuli would be abnormal. To test this hypothesis, behaviors related to sensory aspects of the stress response were quantified in nine groups of mice, aged 4-14 months, comparing knockout with their wild-type counterparts in six distinctly different behavioral tests. Resulting data indicated the following statistically significant differences: open field grooming frequency following saline injection, wild-type > knockout; paw stimulation with Von Frey fibers, knockout < wild-type; balance beam, knockout mobility < WT; thermal sensitivity to heat (tail flick), knockout < wild-type; and cold, knockout < wild-type. Insignificant differences in hanging wire test indicate that these responses are unrelated to reduced muscle strength. Each of these disparate environmental stimuli provided data indicating alterations of responses in knockout mice that suggest participation of osteocalcin in transmission of information about those sensory stimuli.     

Hexacosylferulate,a phenolic constituent of Pinus roxburghii

The occurrence of hexacosylferulate in Pinus roxburghii is reported. Its structure has been derived from spectral measurements, chemical reactions and finally from an unambiguous synthesis.     

Development of an Ointment Formulation Using Hot-Melt Extrusion Technology

Ointments are generally prepared either by fusion or by levigation methods. The current study proposes the use of hot-melt extrusion (HME) processing for the preparation of a polyethylene glycol base ointment. Lidocaine was used as a model drug. A modified screw design was used in this process, and parameters such as feeding rate, barrel temperature, and screw speed were optimized to obtain a uniform product. The product characteristics were compared with an ointment of similar composition prepared by conventional fusion method. The rheological properties, drug release profile, and texture characteristics of the hot-melt extruded product were similar to the conventionally prepared product. This study demonstrates a novel application of the hot-melt extrusion process in the manufacturing of topical semi-solids.     

Application of a Mass Spectrometric Approach to Detect the Presence of Fatty Acid Biosynthetic Phosphopeptides

The details of plant lipid metabolism are relatively well known but the regulation of fatty acid production at the protein level is still not understood. Hence this study explores the importance of phosphorylation as a mechanism to control the activity of fatty acid biosynthetic enzymes using low and high oleic acid mesocarps of oil palm fruit (Elaeis guineensis variety of Tenera). Adaptation of neutral loss-triggered tandem mass spectrometry and selected reaction monitoring to detect the neutral loss of phosphoric acid successfully found several phosphoamino acid-containing peptides. These peptides corresponded to the peptides from acetyl-CoA carboxylase and 3-enoyl-acyl carrier protein reductase as identified by their precursor ion masses. These findings suggest that these enzymes were phosphorylated at 20th week after anthesis. Phosphorylation could have reduce their activities towards the end of fatty acid biosynthesis at ripening stage. Implication of phosphorylation in the regulation of fatty acid biosynthesis at protein level has never been reported.