Generation of highly selective monoclonal antibodies inhibiting a recalcitrant protease using decoy designs

Matrix metalloproteinase-12 (MMP-12), also known as macrophage elastase, is a potent inflammatory mediator and therefore an important pharmacological target. Clinical trial failures of broad-spectrum compound MMP inhibitors suggested that specificity is the key for a successful therapy. To provide the required selectivity, monoclonal antibody (mAb)-based inhibitors are on the rise. However, poor production of active recombinant human MMP-12 catalytic domain (cdMMP-12) presented a technical hurdle for its inhibitory mAb development. We hypothesized that this problem could be solved by designing an expression-optimized cdMMP-12 mutant without structural disruptions at its reaction cleft and surrounding area, and thus isolated active-site inhibitory mAbs could maintain their binding and inhibition functions toward wild-type MMP-12. We combined three advances in the field—PROSS algorithm for cdMMP-12 mutant design, convex paratope antibody library construction, and functional selection for inhibitory mAbs. As a result, isolated Fab inhibitors showed nanomolar affinity and potency toward cdMMP-12 with high selectivity and high proteolytic stability. Particularly, Fab LH11 targeted the reaction cleft of wild-type cdMMP-12 with 75 nM binding K_D and 23 nM inhibition IC₅₀. We expect that our methods can promote the development of mAbs inhibiting important proteases, many of which are recalcitrant to functional production.     

Biochemical changes in the corpus luteum of intrauterine device fitted goats

Parous goats were sham operated (6 goats), bilaterally fitted with IUDs (7 goats), or unilaterally fitted with an IUD isilateral (6 goats) or contralateral (5 goats) to the corpus luteum (CL). Autopsy was on Day 3 or Day 5 of the second or third estrus. The cycle length of IUD-fitted goats was shorter than in controls (6.2 days vs. 16.7 days, p less than .05). Mean CL weight in the contralateral group, 164.8 mg, exceeded mean weight of the ipsilateral group, 92.4 mg (P less than .01). The concentration of total cholesterol was higher in the ipsilateral group than in the contralateral group (3.16 vs. 2.12 mg/gm CL, p less than .01). The protein concentration in CL from the control, unilateral and bilateral groups was 14.4, 12.2, and 23.9 mg, respectively, p less than .05. The content and concentration of progesterone, and total and free cholesterol was higher in sham operated controls than in IUD-fitted goats (not significant). Protein, RNA, and DNA increased considerably in IUD-fitted goats (not significant), indicating a derangement of a luteotrophic rather than luteomorphic mechanism in CL from IUD-fitted goats.     

The Response of Triticum aestivum Treated with Plant Growth Regulators to Acute Day/Night Temperature Rise

The fluctuation in temperature adversely affects grain development when the climate changes intermittently. This study investigated the effect of high day/night temperatures (34/30 °C, 38/34 °C and 42/38 °C) for two stress durations (24 h and 48 h) on Triticum aestivum. To ascertain the role of plant growth regulator (PGR) in alleviating the deleterious effects of high temperature stress, the combination of various PGRs (e.g., methyl jasmonate, salicylic acid, ascorbic acid, calcium chloride and indole acetic acid) were foliar sprayed twice; one week prior to commencement of anthesis stage and immediately after the exposure to high temperature stress. In general, the high temperature reduces plant growth, grain setting, and 100-grain weight. High temperature stress causes deterioration of plant photosynthetic machinery through a significant decline in energy dissipation, linear electron flow (LEF) and quantum yield of photosystem II (Phi2) which led to plant death. An increase in the antioxidant enzymes activity (SOD, APX, and CAT) was observed at 38/34 °C, while their activity declined sharply at 42/38 °C. Grain setting and filling were completely inhibited in plants exposed to 42/38 °C even when treated with different combinations of PGRs. Salicylic acid along with methyl jasmonate was the most effective PGR combination resulting in significant improvements in Phi2, NPQt, SOD, grain filling and grain protein content under high temperature stress. A strong correlation was observed between LEF and chlorophyll contents against the number of grains per spike and 100-grain weight. In summary, acute day and night temperature stress adversely affected wheat morphological, physiological, and yield traits, while foliar application of PGRs was partially effective in mitigating these harmful changes.

     

Isatin-derived Antibacterial and Antifungal Compounds and their Transition Metal Complexes

A series of isatins incorporating thiazole, thiadiazole, benzothiazole and p-toluene sulfonyl hydrazide moieties, along with their cobalt(II), copper(II), nickel(II) and zinc(II) metal complexes have been synthesized and characterized by elemental analyses, molar conductances, magnetic moments, IR, NMR and electronic spectral data. These compounds have been screened for antibacterial activity against Escherichia coli, Bacillus subtillis, Shigella flexneri, Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella typhi, and for antifungal activity against Trichophyton longifusus, Candida albicans, Aspergillus flavus, Microsporum canis, Fusarium solani and Candida glaberata using the agar-well diffusion method. All the synthesized compounds have shown good affinity as antibacterial and/or antifungal agents which increased in most of the cases on complexation with the metal ions.     

Are Two Interviews Better Than One? Eyewitness Memory across Repeated Cognitive Interviews

Eyewitnesses to a filmed event were interviewed twice using a Cognitive Interview to examine the effects of variations in delay between the repeated interviews (immediately & 2 days; immediately & 7 days; 7 & 9 days) and the identity of the interviewers (same or different across the two repeated interviews). Hypermnesia (an increase in total amount of information recalled in the repeated interview) occurred without any decrease in the overall accuracy. Reminiscence (the recall of new information in the repeated interview) was also found in all conditions but was least apparent in the longest delay condition, and came with little cost to the overall accuracy of information gathered. The number of errors, increased across the interviews, but the relative accuracy of participants’ responses was unaffected. However, when accuracy was calculated based on all unique details provided across both interviews and compared to the accuracy of recall in just the first interview it was found to be slightly lower. The identity of the interviewer (whether the same or different across interviews) had no effects on the number of correct details. There was an increase in recall of new details with little cost to the overall accuracy of information gathered. Importantly, these results suggest that witnesses are unlikely to report everything they remember during a single Cognitive Interview, however exhaustive, and a second opportunity to recall information about the events in question may provide investigators with additional information.     

Overcoming biochemical pharmacologic mechanisms of platinum resistance with a texaphyrin-platinum conjugate

In our effort to investigate further texaphyrin conjugation as a means of increasing delivery and accumulation of known anticancer platinum agents in cancer cells, we have continued our studies on the mode of action of a texaphyrin-platinum conjugate, particularly in cisplatin-resistant tumor cells that are characterized by several mechanisms of resistance, including reduced drug accumulation. Our results provide support for the proposal that intracellular platinum and Pt-DNA adduct levels were significantly increased using our conjugate relative to corresponding Pt controls. Moreover, no differences were found in cellular accumulation and Pt-DNA adduct formation between Pt sensitive and Pt resistant ovarian cells. As a result, resistance to the conjugate was lower than cisplatin in resistant cells. Based on these results we conclude that texaphyrin conjugation provides a promising strategy for overcoming biochemical pharmacologic mechanisms of resistance.     

The role of plant-associated rhizobacteria in plant growth,biocontrol and abiotic stress management

The rhizosphere is the region around the plant roots where maximum microbial activities occur. In the rhizosphere, microorganisms' beneficial and harmful activities affect plant growth and development. The mutualistic rhizospheric bacteria which improve plant growth and health are known as plant growth-promoting rhizobacteria (PGPR). They are very important due to their ability to help the plant in diverse ways. PGPR such as Pseudomonas, Bacillus, Azospirillum, Azotobacter, Arthrobacter, Achromobacter, Micrococcus, Enterobacter, Rhizobium, Agrobacterium, Pantoea and Serratia are now very well known. Rhizomicrobiome plays critical roles in nutrient acquisition and assimilation, improved soil texture, secreting and modulating extracellular molecules such as hormones, secondary metabolites, antibiotics and various signal compounds, all leading to the enhancement of plant growth and development. The microbes and compounds they secrete constitute valuable biostimulants and play pivotal roles in modulating plant stress responses. In this review, we highlight the rhizobacteria diversity and cutting-edge findings focusing on the role of a PGPR in plant growth and development. We also discussed the role of PGPR in resisting the adverse effects arising from various abiotic (drought, salinity, heat, heavy metals) stresses.