PLA nanovectors with encapsulated betulin: plant leaf extract-synthesized nanovectors are more efficacious than PVA-synthesized nanovectors

Objectives

Betulin (BT) is an abundant triterpene found predominantly in the bark of Himalayan birch. It is difficult to deliver it in vivo because of its low aqueous solubility. We have therefore developed novel formulations of BT for improving its solubility, bioavailability and therapeutic efficacy.

Results

Poly-d,l-lactide nanovectors (PLA NVs) were synthesized using poly(vinyl alcohol) and Lonicera japonica leaf extract (LE) as a stabiliser and named as PLA-1 NVs and PLA-2 NVs. PLA-1 NVs and PLA-2 NVs were used for the encapsulation of betulin (BT) and named as BT-En-1 and BT-En-2 NVs. The encapsulation efficiency of BT-En-1 and BT-En-2 NVs were 99.3 and 100 % respectively. Prepared nanoformulations were physically stable. An in vitro study revealed 45 % BT was released over 24 h. BT had a prolonged release from BT-En-2 NVs as compared to BT-En-1 NVs. BT-En-2 NVs had better anticancerous activity against SiHa cells than BT-En-1 NVs.

Conclusions

Developed BT-EN-2 NVs had better biocompatibility, excellent stability and enhanced release characteristics than BT-En-1 NVs.

     

Inhibition of galectin-3 mediated cellular interactions by pectic polysaccharides from dietary sources

Pectic polysaccharides from dietary sources such as Decalepis hamiltonii—swallow root (SRPP), Hemidesmus indicus (HPP), Nigella sativa—black cumin (BCPP), Andrographis serpyllifolia—(APP), Zingiber officinale—ginger (GRPP) and, citrus pectin (CPP) were examined for galectin inhibitory activity. Inhibition of (a) galectin-3 of MDA-MB-231 cells induced hemagglutination of red blood cells; (b) galectin-3 mediated interaction between normal/metastatic human buccal cells (NBC)/(MBC) and; (c) invasion of MDA-MB-231 and MBC in the invasive chamber was assessed. Results indicated that SRPP inhibited hemagglutination at Minimum Inhibitory Concentration (MIC) of 1.86 μg ml⁻¹ equivalent of carbohydrate as apposed to those of BCPP (130 μg ml⁻¹), APP (40 μg ml⁻¹), HPP (40 μg ml⁻¹) and CPP (25 μg ml⁻¹). GRPP even at concentration >1–6 mg ml⁻¹ did not inhibit agglutination. Also SRPP showed ∼15 and 2 fold potent anti hemagglutination activity relative to that of galectin-3 specific sugars—galactose (MIC-27.1 μg ml⁻¹) and lactose (MIC-4.16 μg ml⁻¹) respectively. Further, SRPP at 10 μg ml⁻¹ inhibited agglutination of NBC by galectin-3 of MDA-MB-231 cells. Modified swallow root pectic polysaccharide (MSRPP) of 50 kDa retained anti hemagglutination activity (MIC of 1.03 μg ml⁻¹) and inhibited MDA-MB-231 and MBC invasion by 73 and 50% with an IC₅₀ of 136 and 200 μg ml⁻¹ respectively. Both SRPP and MSRPP induced apoptosis up to 80% at 100 μg ml⁻¹ concentration by activating ∼2 and 8 folds of Caspase-3 activity. Sugar composition analysis and its correlation with the galectin inhibitory property indicated that pectic polysaccharides with higher arabinose and galactose content—arabinogalactan inhibited hemagglutination significantly.     

ABL/BCR gene variant with two-step mechanism: Unusual localization and rare/novel chromosomal rearrangements in CML patients

Aims: Variant translocations involving 9q, 22q and at least one additional genomic locus occur in 5-10% of the patients with chronic myeloid leukemia (CML). The mechanisms for the formation of these variant translocations are not fully characterized. Here we report CML cases presenting a variant translocation indicating two-step mechanism with rare/novel chromosomal rearrangement. Methods: Karyotype analysis was performed on metaphases obtained through short-term cultures of bone marrow and blood. Detection of BCR-ABL fusion gene was performed using dual-color dual-fusion (D-FISH) and extra signal (ES) translocation probes. BAC-FISH was also carried out. Results: In Patient 1, the third partner chromosome was der(11)(p15) with a 2F2G1R signal pattern, which is an unusual signal pattern with the two-step mechanism. Patients 2 and 3 showed typical positive (2F1G1R) signal pattern. In Patient 2, both the chromosome 22s were involved in variant formation. The second fusion was observed below the BCR gene of the second homologue. In Patient 3 the third chromosome was der(13)(q14). The fourth patient showed a variant pattern with BCR/ABL-ES probe involving der(X)(q13) region. Conclusion: The presence of different rearrangements of both 9q34 and 22q11 regions highlights the genetic heterogeneity of this subgroup of CML. In each case with variants, further studies with FISH, BAC-FISH or more advanced technique such as microarray should be performed. Future studies should be performed to confirm the presence of true breakpoint hot spots and assess their implications in CML with variant Ph.     

A Hump-Backed Trend in Bacterial Diversity with Elevation on Mount Fuji, Japan

Little is known of how bacterial diversity in soils varies with elevation. One previous study found a decline with elevation, whereas another found no trend. We chose Mount Fuji of Japan as a geologically and topographically simple mountain system. Samples were taken at elevational intervals, between the base of the mountain at 1,000 m and its summit at 3,700 m. Polymerase chain reaction-amplified soil DNA for the bacterial 16S gene targeting V1–V3 region was pyrosequenced using the 454 Roche machine, and taxonomically classified with reference to a bioinformatic database. There was a significant “peak” in total bacterial diversity at around 2,500 m above the tree line with a decline towards the highest elevations around 3,700 m near the summit. Individual bacterial phyla show distinct trends—increase, decrease, or a mid-elevational “bulge” in diversity. Bacterial diversity does not parallel woody plant or herbaceous plant diversity. We suggest that beyond the tree and vegetation line, the more extreme temperature fluctuations, stronger UV, lack of nutrients, and more frequent disturbance of the loose substrate of these slopes allows less competition and greater bacterial species diversity due to “lottery” recruitment. However, at the highest elevations, the physiological challenges are so extreme that fewer bacterial species are capable of surviving.     

Pelvic Floor Dysfunction in Morbidly Obese Women: Pilot Study

Objective: The objective of this study was to evaluate the impact of obesity on pelvic floor function in women. Research Methods and Procedures: This was a prospective controlled study of 20 morbidly obese female patients planning to undergo gastric bypass surgery and 20 age‐matched female controls. Subjects completed symptom and impact questionnaires, including the Incontinence Impact Questionnaire (IIQ‐7), Urogenital Distress Inventory (UDI), the Kobashi Prolapse Symptom Inventory and Quality‐of‐Life Questionnaire (PSI‐QOL), and Index of Female Sexual Function. Data were analyzed with Wilcoxon or ratio χ² tests. Results: Mean weight was 295.7 ± 87.9 lbs in the study group and 144.79 ± 33.07 lbs in the control group. Mean BMI was 52.65 ±14.49 kg/m² in the study group and 25.11 ± 5.27 kg/m² in the control group. According to the IIQ‐7, urinary incontinence significantly affected lifestyle in the study group. The total IIQ‐7 score was also significantly affected in the study group (p = 0.03). The UDI indicated more urinary leakage with activity (p = 0.04) and more incidents of small amounts of leakage (p = 0.02) in the study group. According to the PSI‐QOL, women in the study group experienced constipation more often because of difficulty in emptying the rectum (p = 0.04). The PSI‐QOL score was higher in the study group (6.75 ± 6.84) than in the control group (2.65 ± 3.03; p = 0.04). There were no significant differences between groups regarding sexual function. Discussion: Morbid obesity is associated with a significant negative impact on urogenital health. Sexual function did not seem to be affected in women who are morbidly obese.     

Chemical Composition,Cytotoxic and Antibacterial Activities of Essential Oils of Cultivated Clones of Juniperus communis and Wild Juniperus Species

Needles of seven cultivated clones (C1 – C7) of Juniperus communis at lower altitude and three wild Juniperus species (J. communis, J. recurva and J. indica) at higher altitudes were investigated comparatively for their essential oils (EOs) yields, chemical composition, cytotoxic and antibacterial activities. The EOs yields varied from 0.26 to 0.56% (v/w) among samples. Sixty‐one volatile components were identified by gas chromatography‐mass spectrometry (GC/MS) and quantified using gas chromatography GC (FID) representing 82.5 – 95.7% of the total oil. Monoterpene hydrocarbons (49.1 – 82.8%) dominated in all samples (α‐pinene, limonene and sabinene as major components). Principal component analysis (PCA) of GC data revealed that wild and cultivated Juniperus species are highly distinct due to variation in chemical composition. J. communis (wild species) displayed cytotoxicity against SiHa (human cervical cancer), A549 (human lung carcinoma) and A431 (human skin carcinoma) cells (66.4 ± 2.2%, 74.4 ± 1.4% and 57.4 ± 4.0%), respectively, at 200 μg/ml. EOs exhibited better antibacterial activity against Gram‐positive bacteria than against Gram‐negative bacteria with the highest zone of inhibition against Staphylococcus aureus MTCC 96 (19.2 ± 0.7) by clone‐7. As per the conclusion of the findings, EOs of clone‐2, clone‐5 and clone‐7 can be suggested to the growers of lower altitude, as there is more possibility of uses of these EOs in food and medicinal preparations.     

Promoter polymorphism MMP-1 (-1607 2G/1G) and MMP-3 (-1612 5A/6A) in development of HAND and modulation of pathogenesis of HAND

The pathogenesis of HIV-associated neurocognitive disorder (HAND) is modulated by host genetic susceptibility factors such as Matrix metalloproteinases (MMPs). Promoter polymorphism of MMP-1 and MMP-3 may modify the expression of the gene. Hence, we evaluated the association of MMP-1-16072G/1G and MMP-3-1612 5A/6A polymorphisms with development of HAND and the modulation of pathogenesis of HAND. We enrolled a total of 180 individuals, 50 HIV-infected individuals with HAND, 130 without HAND, and 150 healthy controls. Polymorphism of MMP-1 and MMP-3 were genotyped by PCR-RFLP. MMP-1-1607 2G1G, -16071G/2G-1G/1G genotypes and -1607 1G allele were associated with the development of HAND (OR = 1.64, P = 0.05; OR = 1.45, P = 0.04; OR = 1.69, P = 0.05). MMP-1-16071G1G, MMP-3-16125A5A genotypes increased the risk for the development of HAND (OR = 1.78, P = 0.25; OR = 2.39, P = 0.13). MMP-3-1612 5A5A, -1612 6A/5A-5A/5A genotypes and -1612 5A allele were associated with the reduced risk of HAND (OR = 0.40, P = 0.05; OR = 0.53, P = 0.04; OR = 0.40, P = 0.01). Haplotype 5A1G increased the risk of development of HAND (OR = 1.93, P = 0.05). As observed in advanced HIV disease stage, MMP-1-1607 1G1G genotype enhance the risk for advancement of HIV disease (OR = 1.69, P = 0.89). MMP-3-1612 6A5A genotype showed higher risk for development of HAND in alcohol users (0R = 1.65, P = 0.44). MMP-1 genotype may have an influence on development of HAND whereas MMP3-1612 5A5A genotype may reduce risk for pathogenesis of HAND.     

Countercurrent tangential chromatography for large-scale protein purification

Recent advances in cell culture technology have created significant pressure on the downstream purification process, leading to a "downstream bottleneck" in the production of recombinant therapeutic proteins for the treatment of cancer, genetic disorders, and cardiovascular disease. Countercurrent tangential chromatography overcomes many of the limitations of conventional column chromatography by having the resin (in the form of a slurry) flow through a series of static mixers and hollow fiber membrane modules. The buffers used in the binding, washing, and elution steps flow countercurrent to the resin, enabling high-resolution separations while reducing the amount of buffer needed for protein purification. The results obtained in this study provide the first experimental demonstration of the feasibility of using countercurrent tangential chromatography for the separation of a model protein mixture containing bovine serum albumin and myoglobin using a commercially available anion exchange resin. Batch uptake/desorption experiments were used in combination with critical flux data for the hollow fiber filters to design the countercurrent tangential chromatography system. A two-stage batch separation yielded the purified target protein at >99% purity with 94% recovery. The results clearly demonstrate the potential of using countercurrent tangential chromatography for the large-scale purification of therapeutic proteins.