Effects of cyclophosphamide on pulmonary function in patients with scleroderma and interstitial lung disease: a systematic review and meta-analysis of randomized controlled trials and observational prospective cohort studies

Introduction

The purpose of the present study was to systematically review the effect of cyclophosphamide treatment on pulmonary function in patients with systemic sclerosis and interstitial lung disease.

Methods

The primary outcomes were the mean change in forced vital capacity and in diffusing capacity for carbon monoxide after 12 months of therapy in patients treated with cyclophosphamide.

Results

Three randomized clinical trials and six prospective observational studies were included for analysis. In the pooled analysis, the forced vital capacity and the diffusing capacity for carbon monoxide predicted values after 12 months of therapy were essentially unchanged, with mean changes of 2.83% (95% confidence interval = 0.35 to 5.31) and 4.56% (95% confidence interval = -0.21 to 9.33), respectively.

Conclusions

Cyclophosphamide treatment in patients with systemic sclerosis-related interstitial lung disease does not result in clinically significant improvement of pulmonary function.     

The choice of controls in a case-control study on WBC-DNA adducts and metabolic polymorphisms

The choice of the control group is a key issue in case-control studies, particularly in studies of molecular epidemiology. We discuss the potential bias introduced by different options. To exemplify the consequences of different choices, we have analysed two sets of controls in the context of a case-control study on bladder cancer: 55 were patients with urological conditions (cystitis, prostate hypertrophy), while 49 had a miscellany of medical or surgical conditions. We measured DNA adducts in white blood cells (WBC) by ³²P-postlabelling and a series of metabolic polymorphisms (GSTM1, GSTT1, GSTP1, NAT2, NQO1). While no statistically significant differences were found for metabolic polymorphisms, the two series of controls showed different concentrations of DNA adducts, suggesting that conditions related to bladder cancer or intermediate steps leading to bladder cancer, such as chronic cystitis, may be associated with higher adduct levels. An association between DNA adduct levels and infection has been noted before in experimental animals: both in lung and in the skin, an inflammatory response increased the biologically effective doses of polycyclic aromatic hydrocarbons. An alternative explanation is confounding; in fact, after adjustment for the level of consumption of fruit and vegetables (but not for smoking) the difference between the two control groups was no longer statistically significant. In conclusion, the choice of controls in studies of molecular epidemiology has subtle methodological implications, including confounding of metabolic/molecular measurements by complex exposures such as diet.     

The Salmonella enterica ZinT structure,zinc affinity and interaction with the high-affinity uptake protein ZnuA provide insight into the management of periplasmic zinc

Background

In Gram-negative bacteria the ZnuABC transporter ensures adequate zinc import in Zn(II)-poor environments, like those encountered by pathogens within the infected host. Recently, the metal-binding protein ZinT was suggested to operate as an accessory component of ZnuABC in periplasmic zinc recruitment. Since ZinT is known to form a ZinT–ZnuA complex in the presence of Zn(II) it was proposed to transfer Zn(II) to ZnuA. The present work was undertaken to test this claim.

Methods

ZinT and its structural relationship with ZnuA have been characterized by multiple biophysical techniques (X-ray crystallography, SAXS, analytical ultracentrifugation, fluorescence spectroscopy).

Results

The metal-free and metal-bound crystal structures of Salmonella enterica ZinT show one Zn(II) binding site and limited structural changes upon metal removal. Spectroscopic titrations with Zn(II) yield a K_D value of 22 ± 2 nM for ZinT, while those with ZnuA point to one high affinity (K_D < 20 nM) and one low affinity Zn(II) binding site (K_D in the micromolar range). Sedimentation velocity experiments established that Zn(II)-bound ZinT interacts with ZnuA, whereas apo-ZinT does not. The model of the ZinT–ZnuA complex derived from small angle X-ray scattering experiments points to a disposition that favors metal transfer as the metal binding cavities of the two proteins face each other.

Conclusions

ZinT acts as a Zn(II)-buffering protein that delivers Zn(II) to ZnuA.

General significance

Knowledge of the ZinT–ZnuA relationship is crucial for understanding bacterial Zn(II) uptake.     

Seasonality Modifies Methylation Profiles in Healthy People

DNA methylation is a well-characterized epigenetic modification that plays an important role in the regulation of gene expression. There is growing evidence on the involvement of epigenetic mechanisms in disease onset, including cancer. Environmental factors seem to induce changes in DNA methylation affecting human health. However, little is known about basal methylation levels in healthy people and about the correlation between environmental factors and different methylation profiles. We investigated the effect of seasonality on basal methylation by testing methylation levels in the long interspersed nucleotide element-1 (LINE-1) and in two cancer-related genes (RASSF1A and MGMT) of 88 healthy male heavy smokers involved in an Italian randomized study; at enrolment the subjects donated a blood sample collected in different months. Methylation analyses were performed by pyrosequencing. Mean methylation percentage was higher in spring and summer for the LINE1, RASSF1A and MGMT genes (68.26%, 2.35%, and 9.52% respectively) compared with autumn and winter (67.43%, 2.17%, and 8.60% respectively). In particular, LINE-1 was significantly hypomethylated (p = 0.04 or 0.05 depending on the CpG island involved) in autumn and winter compared with spring and summer. Seasonality seems to be a modifier of methylation levels and this observation should be taken into account in future analyses.     

Mitochondrial Ca(2+) and apoptosis

Mitochondria are key decoding stations of the apoptotic process. In support of this view, a large body of experimental evidence has unambiguously revealed that, in addition to the well-established function of producing most of the cellular ATP, mitochondria play a fundamental role in triggering apoptotic cell death. Various apoptotic stimuli cause the release of specific mitochondrial pro-apoptotic factors into the cytosol. The molecular mechanism of this release is still controversial, but there is no doubt that mitochondrial calcium (Ca(2+)) overload is one of the pro-apoptotic ways to induce the swelling of mitochondria, with perturbation or rupture of the outer membrane, and in turn the release of mitochondrial apoptotic factors into the cytosol. Here, we review as different proteins that participate in mitochondrial Ca(2+) homeostasis and in turn modulate the effectiveness of Ca(2+)-dependent apoptotic stimuli. Strikingly, the final outcome at the cellular level is similar, albeit through completely different molecular mechanisms: a reduced mitochondrial Ca(2+) overload upon pro-apoptotic stimuli that dramatically blunts the apoptotic response.     

Good design practices for an integrated containment and production system for advanced therapies

Advances in molecular biology and the possibility of differentiating stem cells have opened up new scenarios in therapies that use progenitor or variously differentiated cells. Regardless of the choice of the system, designing a plant for producing advanced therapies requires a clear understanding of the final objective (the product), taking into account all the regulatory, environment, process, risk assessment, asepsis, and validation aspects involved until its implementation. Good Manufacturing Practice (GMP) compliant procedures are a prerequisite for cell production in clinical application, and clean rooms are zones for producing cell therapies. Clean rooms for clinical application require high running and maintenance costs and need trained operators and strict procedures to prepare the rooms and the people involved in the processes. While today production mainly occurs in open systems (clean rooms), there is evidence of processes in closed systems (isolators). The isolator is a Grade A aseptic closed system that requires a controlled environment and at least a Grade D environment in the case of sterile productions (A in D closed system). The use of isolators can ensure a very high level of protection against the risk of product contamination and, at the same time, provide the operators with a very safe working environment. Furthermore, working with closed systems can optimize and facilitate the production of Advanced Therapy Medical Products in GMP environments, by providing an easily reproducible working tool even for large-scale production, with generally lower costs compared to a classical clean room approach. In conclusion, the isolator workstation as a possible alternative to the classic clean room, due to its small size and the simplification of the working and maintenance operational procedures, may represent an interesting solution in the perspective of the increasingly more stringent requests for cost reductions of GMP in clinical application.     

Population genomics reveals evolution and variation of Saccharomyces cerevisiae in the human and insects gut

The quest to discover the variety of ecological niches inhabited by Saccharomyces cerevisiae has led to research in areas as diverse as wineries, oak trees and insect guts. The discovery of fungal communities in the human gastrointestinal tract suggested the host's gut as a potential reservoir for yeast adaptation. Here, we report the existence of yeast populations associated with the human gut (HG) that differ from those isolated from other human body sites. Phylogenetic analysis on 12 microsatellite loci and 1715 combined CDSs from whole-genome sequencing revealed three subclusters of HG strains with further evidence of clonal colonization within the host's gut. The presence of such subclusters was supported by other genomic features, such as copy number variation, absence/introgressions of CDSs and relative polymorphism frequency. Functional analysis of CDSs specific of the different subclusters suggested possible alterations in cell wall composition and sporulation features. The phenotypic analysis combined with immunological profiling of these strains further showed that sporulation was related with strain-specific genomic characteristics in the immune recognition pattern. We conclude that both genetic and environmental factors involved in cell wall remodelling and sporulation are the main drivers of adaptation in S. cerevisiae populations in the human gut.     

The crystal structure of the sorcin calcium binding domain provides a model of Ca2+-dependent processes in the full-length protein

Sorcin is a 21.6 kDa calcium binding protein, expressed in a number of mammalian tissues that belongs to the small, recently identified penta-EF-hand (PEF) family. Like all members of this family, sorcin undergoes a Ca2+-dependent translocation from cytosol to membranes where it binds to target proteins. For sorcin, the targets differ in different tissues, indicating that it takes part in a number of Ca2+-regulated processes. The sorcin monomer is organized in two domains like in all PEF proteins: a flexible, hydrophobic, glycine-rich N-terminal region and a calcium binding C-terminal domain. In vitro, the PEF proteins are dimeric in their Ca2+-free form, but have a marked tendency to precipitate when bound to calcium. Stabilization of the dimeric structure is achieved by pairing of the uneven EF-hand, EF5. Sorcin can also form tetramers at acid pH.The sorcin calcium binding domain (SCBD, residues 33-198) expressed in Escherichia coli was crystallized in the Ca2+-free form. The structure was solved by molecular replacement and was refined to 2.2 A with a crystallographic R-factor of 22.4 %. Interestingly, the asymmetric unit contains two dimers.The structure of the SCBD leads to a model that explains the solution properties and describes the Ca2+-induced conformational changes. Phosphorylation studies show that the N-terminal domain hinders phosphorylation of SCBD, i.e. the rate of phosphorylation increased twofold in the absence of the N-terminal region. In addition, previous fluorescence studies indicated that hydrophobic residues are exposed to solvent upon Ca2+ binding to full-length sorcin. The model accounts for these data by proposing that Ca2+ binding weakens the interactions between the two domains and leads to their reorientation, which exposes hydrophobic regions facilitating the Ca2+-dependent binding to target proteins at or near membranes.