The Suitability of P. falciparum Merozoite Surface Proteins 1 and 2 as Genetic Markers for In Vivo Drug Trials in Yemen

Background

The accuracy of the conclusions from in vivo efficacy anti-malarial drug trials depends on distinguishing between recrudescences and re-infections which is accomplished by genotyping genes coding P. falciparum merozoite surface 1 (MSP1) and MSP2. However, the reliability of the PCR analysis depends on the genetic markers’ allelic diversity and variant frequency. In this study the genetic diversity of the genes coding for MSP1 and MSP2 was obtained for P. falciparum parasites circulating in Yemen.

Methods

Blood samples were collected from 511 patients with fever and screened for malaria parasites using Giemsa-stained blood films. A total 74 samples were infected with P. falciparum, and the genetic diversity was assessed by nested PCR targeting Pfmsp1 (Block2) and Pfmsp2 (block 3).

Results

Overall, 58%, 28% and 54% of the isolates harboured parasites of the Pfmsp1 K1, MAD20 and RO33 allelic families, and 55% and 89% harboured those of the Pfmsp2 FC27 and 3D7 allelic families, respectively. For both genetic makers, the multiplicity of the infection (MOI) was significantly higher in the isolates from the foothills/coastland areas as compared to those from the highland (P<0.05). Pfmsp2 had higher number of distinct allelic variants than Pfmsp1 (20 vs 11). The expected heterozygosity (HE) for Pfmsp1 and Pfmsp2 were 0.82 and 0.94, respectively. Nonetheless, a bias in the frequency distribution of the Pfmsp1 allelic variants was noted from all areas, and of those of Pfmsp2 in the samples collected from the highland areas.

Conclusions

Significant differences in the complexity and allelic diversity of Pfmsp1 and Pfmsp2 genes between areas probably reflect differences in the intensity of malaria transmission. The biased distribution of allelic variants suggests that in Yemen Pfmsp1 should not be used for PCR correction of in vivo clinical trials outcomes, and that caution should be exercised when employing Pfmsp2.     

Pseudoxanthoma Elasticum: Cardiac Findings in Patients and Abcc6-Deficient Mouse Model

Background

Pseudoxanthoma elasticum (PXE), caused by mutations in the ABCC6 gene, is a rare multiorgan disease characterized by the mineralization and fragmentation of elastic fibers in connective tissue. Cardiac complications reportedly associated with PXE are mainly based on case reports.

Methods

A cohort of 67 PXE patients was prospectively assessed. Patients underwent physical examination, electrocardiogram, transthoracic echocardiography, cardiac magnetic resonance imaging (CMR), treadmill testing, and perfusion myocardial scintigraphy (SPECT). Additionally, the hearts of a PXE mouse models (Abcc6^−/−) and wild-type controls (WT) were analyzed.

Results

Three patients had a history of proven coronary artery disease. In total, 40 patients underwent exercise treadmill tests, and 28 SPECT. The treadmill tests were all negative. SPECT showed mild perfusion abnormalities in two patients. Mean left ventricular (LV) dimension and function values were within the normal range. LV hypertrophy was found in 7 (10.4%) patients, though the hypertrophy etiology was unknown for 3 of those patients. Echocardiography revealed frequent but insignificant mitral and tricuspid valvulopathies. Mitral valve prolapse was present in 3 patients (4.5%). Two patients exhibited significant aortic stenosis (3.0%). While none of the functional and histological parameters diverged significantly between the Abcc6^−/− and WT mice groups at age of 6 and 12 months, the 24-month-old Abcc6^−/− mice developed cardiac hypertrophy without contractile dysfunction.

Conclusions

Despite sporadic cases, PXE does not appear to be associated with frequent cardiac complications. However, the development of cardiac hypertrophy in the 24-month-old Abcc6^−/− mice suggests that old PXE patients might be prone to developing late cardiopathy.     

Determinants of Microdamage in Elderly Human Vertebral Trabecular Bone

Previous studies have shown that microdamage accumulates in bone as a result of physiological loading and occurs naturally in human trabecular bone. The purpose of this study was to determine the factors associated with pre-existing microdamage in human vertebral trabecular bone, namely age, architecture, hardness, mineral and organic matrix. Trabecular bone cores were collected from human L2 vertebrae (n = 53) from donors 54–95 years of age (22 men and 30 women, 1 unknown) and previous cited parameters were evaluated. Collagen cross-link content (PYD, DPD, PEN and % of collagen) was measured on surrounding trabecular bone. We found that determinants of microdamage were mostly the age of donors, architecture, mineral characteristics and mature enzymatic cross-links. Moreover, linear microcracks were mostly associated with the bone matrix characteristics whereas diffuse damage was associated with architecture. We conclude that linear and diffuse types of microdamage seemed to have different determinants, with age being critical for both types.     

HCMV Activates the IL-6-JAK-STAT3 Axis in HepG2 Cells and Primary Human Hepatocytes

Objectives

There has been increased interest in the possible role of human cytomegalovirus (HCMV) in carcinogenesis during the last decade. HCMV seroprevalence was enhanced in patients with hepatocellular carcinoma (HCC) but a possible relationship between HCC and HCMV infection remained to be assessed. The aim of this work was to investigate the pro-tumor influence of HCMV on primary human hepatocytes (PHH) and HepG2 cells.

Methods

Following infection of PHH and HepG2 cells by two different strains of HCMV, we measured the production of IL-6 in culture supernatants by ELISA and the protein levels of STAT3, pSTAT3, JAK, cyclin D1, survivin, p53, p21, and Mdm2 by western Blotting in infected and uninfected cells. Cell proliferation and transformation were investigated using Ki67Ag expression measurement and soft-agar colony formation assay respectively.

Results

Infection of HepG2 cells and PHH by HCMV resulted in the production of IL-6 and the subsequent activation of the IL-6R-JAK-STAT3 pathway. HCMV increased the expression of cyclin D1 and survivin. Cell proliferation was enhanced in HepG2 and PHH infected with HCMV, despite a paradoxical overexpression of p53 and p21. More importantly, we observed the formation of colonies in soft agar seeded with PHH infected with HCMV and when we challenged the HepG2 cultures to form tumorspheres, we found that the HCMV-infected cultures formed 2.5-fold more tumorspheres than uninfected cultures.

Conclusion

HCMV activated the IL-6-JAK-STAT3 pathway in PHH and HepG2 cells, favored cellular proliferation, induced PHH transformation and enhanced HepG2 tumorsphere formation. Our observations raise the possibility that HCMV infection might be involved in the genesis of hepatocellular carcinoma.     

Internal ethnicity in the ethnic economy

Internal ethnicity refers to ethnic subgroups within an immigrant group. An ‘ethnic economy’ includes the self‐employed and their co‐ethnic workers. Although most research treats the boundaries of ‘ethnic economy’ and its variant, the ‘ethnic enclave economy’, as though they were coterminous with those of national‐origin immigrant groups, this assumption is unreliable. Ethnic boundaries need not coincide with those of nationality origin when internal ethnicity exists. To test this hypothesis, we utilize survey data collected from a sample of Iranians in Los Angeles. Because this national‐origin immigrant group contains four ethno‐religious subgroups (Armenians, Bahais, Jews and Muslims), the Iranians in Los Angeles operated four distinctive ethnic economies, not one. Each ethno‐religious subgroup had its own ethnic economy, and these separate economies were only weakly tied to an encompassing Iranian ethnic economy.     

Formation of Hydroxysulphate Green Rust 2 as a Single Iron(II-III) Mineral in Microbial Culture

Although GR2(SO₄ ^2-) can be easily formed by abiotic synthesis, the biotic formation of hydroxysulphate as a single iron(II-III) mineral in microbial culture and its characterization was not achieved. This study was carried out to investigate the sole formation of GR2(SO₄ ^2-) during the reduction of γ-FeOOH by a dissimilatory iron-respiring bacterium, Shewanella putrefaciens CIP 8040^T. Reduction experiments were performed in a non-buffered medium devoid of organic compounds, with 25 mM of sulphate and with a range of lepidocrocite concentrations with H₂ as the electron donor under nongrowth conditions. The resulting biogenic solids, after iron-respiring activity, were characterized by X-ray diffraction (XRD), transmission Mössbauer spectroscopy (TMS) and electron microscopy (SEM and TEM). The sulphate has been identified as the intercalated anion by diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS). In addition, the structure of this sulphate anion was discussed. Our experimental study demonstrated that, under H₂ atmosphere, the biogenic solid was a GR2(SO₄ ^2-), as the sole iron(II-III) bearing mineral, whatever the initial lepidocrocite concentration. The crystals of the biotically formed GR2(SO₄ ^2-) are significantly larger than those observed for GR2(SO₄ ^2-) obtained through abiotic preparation, < 15 μ m diameter as against 0.5–4 μm, respectively.     

Competitive Formation of Hydroxycarbonate Green Rust 1 versus Hydroxysulphate Green Rust 2 in Shewanella putrefaciens Cultures

The formation of hydroxysulphate green rust 2, a Fe(II-III) compound commonly found during corrosion processes of iron-based materials in seawater, has not yet been reported in bacterial cultures. Here we used Shewanella putrefaciens, a dissimilatory iron-reducing bacterium to anaerobically catalyze the transformation of a ferric oxyhydroxide, lepidocrocite (γ-FeOOH), into Fe(II) in the presence of various sulphate concentrations. Biotransformation assays of γ-FeOOH were performed with formate as the electron donor under a variety of concentrations. The results showed that the competitive formation of hydroxycarbonate green rust 1 (GR1(CO₃ ^2?)) and hydroxysulphate green rust 2 (GR2(SO₄ ^{2 ?})) depended upon the relative ratio (R) of bicarbonate and sulphate concentrations. When R ≥ 0.17, GR1(CO₃ ^{2 ?}) only was formed whereas when R < 0.17, a mixture of GR2(SO₄ ^{2 ?}) and GR1(CO₃ ^{2 ?}) was obtained. These results demonstrated that the hydroxysulphate GR2 can originate from the microbial reduction of γ-FeOOH and confirmed the preference for carbonate over sulphate during green rust precipitation. The solid phases were characterized by X-ray diffraction, transmission Mössbauer spectroscopy and scanning electron microscopy. Diffuse reflectance infrared Fourier transform spectroscopy confirmed the presence of intercalated carbonate and sulphate in green rust's structure. This study sheds light on the influence of dissimilatory iron-reducing bacteria on microbiologically influenced corrosion.     

A combinatorial approach to the structure elucidation of a pyoverdine siderophore produced by a Pseudomonas putida isolate and the use of pyoverdine as a taxonomic marker for typing P. putida subspecies

The structure of a pyoverdine produced by Pseudomonas putida, W15Oct28, was elucidated by combining mass spectrometric methods and bioinformatics by the analysis of non-ribosomal peptide synthetase genes present in the newly sequenced genome. The only form of pyoverdine produced by P. putida W15Oct28 is characterized to contain α-ketoglutaric acid as acyl side chain, a dihydropyoverdine chromophore, and a 12 amino acid peptide chain. The peptide chain is unique among all pyoverdines produced by Pseudomonas subspecies strains. It was characterized as –l-Asp-l-Ala-d-AOHOrn-l-Thr-Gly-c[l-Thr(O-)-l-Hse-d-Hya-l-Ser-l-Orn-l-Hse-l-Ser-O-]. The chemical formula and the detected and calculated molecular weight of this pyoverdine are: C₆₅H₉₃N₁₇O₃₂, detected mass 1624.6404 Da, calculated mass 1624.6245. Additionally, pyoverdine structures from both literature reports and bioinformatics prediction of the genome sequenced P. putida strains are summarized allowing us to propose a scheme based on pyoverdines structures as tool for the phylogeny of P. putida. This study shows the strength of the combination of in silico analysis together with analytical data and literature mining in determining the structure of secondary metabolites such as peptidic siderophores.