Ocaratuzumab,an Fc-engineered antibody demonstrates enhanced antibody-dependent cell-mediated cytotoxicity in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is common in both developed and developing nations where the need for inexpensive and convenient administration of therapy is apparent. Ocaratuzumab is a novel Fc-engineered humanized IgG1 anti-CD20 monoclonal antibody (mAb) designed for effective antibody-dependent cell-mediated cytotoxicity (ADCC) at very low concentrations that may facilitate sub-cutaneous (vs. intravenous) dosing. Here, we report ocaratuzumab’s potency against CLL cells. In vitro assessment of ocaratuzumab’s direct cytotoxicity (DC), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), and ADCC was performed on CLL cells. Ocaratuzumab induced DC, CDC, and ADCP similarly to rituximab or ofatumumab (anti-CD20 mAbs). However, ocaratuzumab showed an advantage in NK cell-mediated ADCC over these antibodies. In allogeneic ADCC, [E:T (effector:target) ratios = 25:1, 12:1, 6:1], ocaratuzumab (10 µg/mL) improved ADCC by ~3-fold compared with rituximab or ofatumumab (P < 0.001 all tested E:T ratios). Notably, the superiority of ocaratuzumab-induced ADCC was observed at low concentrations (0.1–10 ug/ml; P < 0.03; allogeneic assays). In extended allogeneic ADCC E:T titration, ocaratuzumab (0.1 µg/mL) demonstrated 19.4% more cytotoxicity than rituximab (E:T = 0.38:1; P = 0.0066) and 21.5% more cytotoxicity than ofatumumab (E:T = 1.5:1; P = 0.0015). In autologous ADCC, ocaratuzumab (10 µg/mL) demonstrated ~1.5-fold increase in cytotoxicity compared with rituximab or ofatumumab at all E:T ratios tested (E:Ts = 25:1,12:1,6:1; all P < 0.001). Obinutuzumab, a glyco-engineered anti-CD20 mAb, showed no improvement in ADCC activity compared with ocaratuzumab. The enhanced ADCC of ocaratuzumab suggests that it may be effective at low concentrations. If supported by clinical investigation, this feature could potentially allow for subcutaneous dosing at low doses that could expand the potential of administering chemoimmunotherapy in developing countries.     

Effect of gibberellic acid on the vase life and oxidative activities in senescing cut gladiolus flowers

Cut flowers face the problem of short display life and lose their aesthetic value rapidly. In order to enhance the vase life of gladiolus, its cut spikes were subjected to different levels of gibberellic acid (GA₃), viz., 0, 25, 50, 100 and 200 mg L^?1 in vase solution, during two consecutive years 2010 and 2011. The GA₃ treatment significantly influenced the vase quality attributes and antioxidants capacity of gladiolus cut flowers. Gibberellic acid at 25 mg L^?1 caused the longest time taken to open the floret and increased the floret opening, vase life duration and fresh weight. The highest antioxidative activities of superoxide dismutase and free radicals scavenging were also recorded with GA₃ at 25 mg L^?1. The highest peroxidase, catalase activity and the lowest membrane leakage were recorded with GA₃ at 50 mg L^?1. Present study concludes that GA₃ applied at lower concentrations (25 mg L^?1) renders greater beneficial effects on vase life quality, membrane stability and antioxidant activities in gladiolus cut spike, and further higher application rates cause no improvement in the flower longevity.     

Autosomal Recessive Transmission of a Rare KRT74 Variant Causes Hair and Nail Ectodermal Dysplasia: Allelism with Dominant Woolly Hair/Hypotrichosis

Pure hair and nail ectodermal dysplasia (PHNED) comprises a heterogeneous group of rare heritable disorders characterized by brittle hair, hypotrichosis, onychodystrophy and micronychia. Autosomal recessive (AR) PHNED has previously been associated with mutations in either KRT85 or HOXC13 on chromosome 12p11.1-q14.3. We investigated a consanguineous Pakistani family with AR PHNED linked to the keratin gene cluster on 12p11.1 but without detectable mutations in KRT85 and HOXC13. Whole exome sequencing of affected individuals revealed homozygosity for a rare c.821T>C variant (p.Phe274Ser) in the KRT74 gene that segregates AR PHNED in the family. The transition alters the highly conserved Phe274 residue in the coil 1B domain required for long-range dimerization of keratins, suggesting that the mutation compromises the stability of intermediate filaments. Immunohistochemical (IHC) analyses confirmed a strong keratin-74 expression in the nail matrix, the nail bed and the hyponychium of mouse distal digits, as well as in normal human hair follicles. Furthermore, hair follicles and epidermis of an affected family member stained negative for Keratin-74 suggesting a loss of function mechanism mediated by the Phe274Ser substitution. Our observations show for the first time that homozygosity for a KRT74 missense variant may be associated with AR PHNED. Heterozygous KRT74 mutations have previously been associated with autosomal dominant woolly hair/hypotrichosis simplex (ADWH). Thus, our findings expand the phenotypic spectrum associated with KRT74 mutations and imply that a subtype of AR PHNED is allelic with ADWH.     

Frequency distribution and association of Fat-mass and obesity (FTO) gene SNP rs-9939609 variant with Diabetes Mellitus Type-II population of Hyderabad,Sindh, Pakistan

Background/AimDiabetes Mellitus (DM) is one of the important public health issues worldwide. The Fat mass obesity (FTO) gene rs-9939609 variant identified single nucleotide polymorphism (SNP) with the T to A missense mutation, and has a strong association with T2DM. FTO gene is present on chromosome “16q12.2” comprising of nine exons. FTO gene rs-9939609 a variant is commonly found in the Pakistani Population. The purpose of the study was to alert the population about the rs-9939609 variant SNP, having a strong association with T2DM.Material and MethodsTotal of 190 participants were included in the present cross-sectional study. To collect the samples non-probability convenience technique was used. subjects were recruited and divided into three groups, normal healthy subjects, obese and T2DM. The patients were selected from the Medicine department Jamshoro/Hyderabad by filling the pre-designed proforma, as well as verbal and written consent taken from study participants. To analysed the data ANOVA Post hoc (Tukey-test) was applied for comparison among groups (P < 0.05) and “SNP-STAT” online software was used for frequencies.ResultsThe BMI, neck circumference, waist circumference and lipid profile, fasting blood sugar and HbA1c was found significant (p < 0.001) in both genders as compared to control. Homozygous and heterozygous distribution of allelic and genotyping frequency was found in study participants. 37.9 %T/A, 57.4% T/T, and A/A were 4.7%. The FTO gene rs-9939609 variant amplified and have an increased risk of developing T2DM in the Sindh population. Codominant model odd ratio of T/A showed 2.42 (CI)1.23–3.84, with significant p < 0.032.ConclusionThe present study concluded that the FTO gene SNP rs-9939609 variant was found in the population of Hyderabad, Sindh and having strong association with T2DM and obese individuals. Increase BMI, neck and waist circumference are the biomarkers of obesity and causative factors of T2DM.     

Characterization of <Emphasis Type="Italic">VvPAL-like</Emphasis> promoter from grapevine using transgenic tobacco plants

A 2000-bp 5′-flanking region of VvPAL-like was isolated from ‘Summer Black’ grapevine by PCR amplification, named pVvPAL-like. To gain a better understanding of the expression and regulatory mechanism of VvPAL-like, a chimeric expression unit consisting of the β-glucuronidase (GUS) reporter gene under the control of a 2000-bp fragment of the VvPAL-like promoter was transformed into tobacco via Agrobacterium tumefaciens. Histochemical staining showed that the full-length promoter directs efficient expression of the reporter gene in cotyledons and hypocotyls, stigma, style, anthers, pollen, ovary, trichomes, and vascular bundles of transgenic plants. A series of 5′ progressive deletions of the promoter revealed the presence of a negative regulatory region (?424 to ?292) in the VvPAL-like promoter. Exposure of the transgenic tobacco plants to various abiotic stresses demonstrated that the full-length construct could be induced by light, copper (Cu), abscisic acid (ABA), indole-3-acetic (IAA), methyl jasmonate (MeJA) (N-1-naphthylphthalamic acid), ethylene, and drought. Furthermore, the ethylene-responsive region was found to be located in the ?1461/?930 fragment, while the element(s) for the MeJA-responsive expression may be present in the ?424/?292 region in the VvPAL-like promoter. These findings will help us to better understand the molecular mechanisms by which VvPAL-like participates in biosynthesis of flavonoids and stress responses.     

Changes in Body Wall of Sea Cucumber (<Emphasis Type="Italic">Stichopus japonicus</Emphasis>) during a two-Step Heating Process Assessed by Rheology,LF-NMR,and Texture Profile Analysis

Sea cucumber (Stichopus japonicus) is regarded as a highly priced delicacy in Japan and China owing to its unique elasticity and palatability. Heating is an essential step to process sea cucumber with desired texture. In the present study, sea cucumber body wall was pre-heated at 40 °C, followed by post-heating at 80 °C. Changes in body wall were assessed by rheology, LF-NMR, and texture profile analysis. The small strain rheological analysis indicated that there were significant structural changes of body wall at about 40 °C during pre-heating. Large strain texture profile analysis and the storage modulus showed a good correlation. LF-NMR analysis displayed only one water population in samples pre-heated at 40 °C for up to 120 min, and the population did not change significantly with the heating duration. This demonstrated that structural changes and moisture loss from the inter collagen fibrils were not significant. As for samples post-heated at 80 °C, three distinct peaks were found and the T ₂ relaxation time and area of the bulk water decreased significantly, revealing significant changes of internal structures and loss of water-holding capacity. T ₂ parameters had a good correlation to texture profile analysis parameters for samples after both pre-heating and post-heating.     

Psychrophilic and psychrotrophic fungi: a comprehensive review

This article reviews the comparative diversity of psychrophilic and psychrotrophic fungi, their adaptability mechanisms for survival and potential applications in biotechnology and pharmaceuticals. Fungi are able to grow and survive at low temperature and exist widely in polar and non-polar habitats. These cold regions are known for very low temperature, high ultra violet-B radiation, frequent freeze and thaw cycles and low water and nutrient availability. Most of the fungi adapt to such harsh conditions by evolving various strategies in their metabolism and physiology. Psychrophilic and psychrotrophic fungi are of importance in biotechnological and pharmaceutical fields due to their diverse characteristics developed or evolved due to their adaptation and survival in extreme environments, like; production of cold-active enzymes, pharmaceutical or bioactive metabolites and exo-polysaccharides, have potential for bioremediation and can also be used as biofertilizer.     

Inhibiting Tyrosine Phosphorylation of Protein Kinase Cδ (PKCδ) Protects the Salivary Gland from Radiation Damage

Radiation therapy for head and neck cancer can result in extensive damage to normal adjacent tissues such as the salivary gland and oral mucosa. We have shown previously that tyrosine phosphorylation at Tyr-64 and Tyr-155 activates PKCδ in response to apoptotic stimuli by facilitating its nuclear import. Here we have identified the tyrosine kinases that mediate activation of PKCδ in apoptotic cells and have explored the use of tyrosine kinase inhibitors for suppression of irradiation-induced apoptosis. We identify the damage-inducible kinase, c-Abl, as the PKCδ Tyr-155 kinase and c-Src as the Tyr-64 kinase. Depletion of c-Abl or c-Src with shRNA decreased irradiation- and etoposide-induced apoptosis, suggesting that inhibitors of these kinases may be useful therapeutically. Pretreatment with dasatinib, a broad spectrum tyrosine kinase inhibitor, blocked phosphorylation of PKCδ at both Tyr-64 and Tyr-155. Expression of “gate-keeper” mutants of c-Abl or c-Src that are active in the presence of dasatinib restored phosphorylation of PKCδ at Tyr-155 and Tyr-64, respectively. Imatinib, a c-Abl-selective inhibitor, also specifically blocked PKCδ Tyr-155 phosphorylation. Dasatinib and imatinib both blocked binding of PKCδ to importin-α and nuclear import, demonstrating that tyrosine kinase inhibitors can inhibit nuclear accumulation of PKCδ. Likewise, pretreatment with dasatinib also suppressed etoposide and radiation induced apoptosis in vitro. In vivo, pre-treatment of mice with dasatinib blocked radiation-induced apoptosis in the salivary gland by >60%. These data suggest that tyrosine kinase inhibitors may be useful prophylactically for protection of nontumor tissues in patients undergoing radiotherapy of the head and neck.