Analysis of Spliceosomal Proteins in Trypanosomatids Reveals Novel Functions in mRNA Processing

In trypanosomatids, all mRNAs are processed via trans-splicing, although cis-splicing also occurs. In trans-splicing, a common small exon, the spliced leader (SL), which is derived from a small SL RNA species, is added to all mRNAs. Sm and Lsm proteins are core proteins that bind to U snRNAs and are essential for both these splicing processes. In this study, SmD3- and Lsm3-associated complexes were purified to homogeneity from Leishmania tarentolae. The purified complexes were analyzed by mass spectrometry, and 54 and 39 proteins were purified from SmD3 and Lsm complexes, respectively. Interestingly, among the proteins purified from Lsm3, no mRNA degradation factors were detected, as in Lsm complexes from other eukaryotes. The U1A complex was purified and mass spectrometry analysis identified, in addition to U1 small nuclear ribonucleoprotein (snRNP) proteins, additional co-purified proteins, including the polyadenylation factor CPSF73. Defects observed in cells silenced for U1 snRNP proteins suggest that the U1 snRNP functions exclusively in cis-splicing, although U1A also participates in polyadenylation and affects trans-splicing. The study characterized several trypanosome-specific nuclear factors involved in snRNP biogenesis, whose function was elucidated in Trypanosoma brucei. Conserved factors, such as PRP19, which functions at the heart of every cis-spliceosome, also affect SL RNA modification; GEMIN2, a protein associated with SMN (survival of motor neurons) and implicated in selective association of U snRNA with core Sm proteins in trypanosomes, is a master regulator of snRNP assembly. This study demonstrates the existence of trypanosomatid-specific splicing factors but also that conserved snRNP proteins possess trypanosome-specific functions.     

Synthesis and in vivo evaluation of [11C]MPTQ: A potential PET tracer for alpha2A-adrenergic receptors

Radiosynthesis and in vivo evaluation of [N-methyl-¹¹C] 5-methyl-3-[4-(3-phenylallyl)-piperazin-1-ylmethyl]-3,3a,4,5-tetrahydroisoxazolo[4,3-c]quinoline (1), a potential PET tracer for alpha2-adrenergic receptors is described. Syntheses of nonradioactive standard 1 and corresponding desmethyl precursor 2 were achieved from 2-aminobenzaldehyde in 40% and 65% yields, respectively. Methylation using [¹¹C]CH₃I in presence of aqueous potassium hydroxide in DMSO afforded [¹¹C]1 in 25% yield (EOS) with >99% chemical and radiochemical purities with a specific activity ranged from 3–4 Ci/μmol (n = 6). The total synthesis time was 30 min from EOB. PET studies in anesthetized baboon show that [¹¹C]1 penetrates BBB and accumulates in alpha2A-AR enriched brain areas.     

Simultaneous Improved Performance and Thermal Stability of Planar Metal Ion Incorporated CsPbI2Br All‐Inorganic Perovskite Solar Cells Based on MgZnO Nanocrystalline Electron Transporting Layer

The high thermal stability and facile synthesis of CsPbI₂Br all‐inorganic perovskite solar cells (AI‐PSCs) have attracted tremendous attention. As far as electron‐transporting layers (ETLs) are concerned, low temperature processing and reduced interfacial recombination centers through tunable energy levels determine the feasibility of the perovskite devices. Although the TiO₂ is the most popular ETL used in PSCs, its processing temperature and moderate electron mobility hamper the performance and feasibility. Herein, the highly stable, low‐temperature processed MgZnO nanocrystal‐based ETLs for dynamic hot‐air processed Mn²⁺ incorporated CsPbI2Br AI‐PSCs are reported. By holding its regular planar “n–i–p” type device architecture, the MgZnO ETL and poly(3‐hexylthiophene‐2,5‐diyl) hole transporting layer, 15.52% power conversion efficiency (PCE) is demonstrated. The thermal‐stability analysis reveals that the conventional ZnO ETL‐based AI‐PSCs show a serious instability and poor efficiency than the Mg²⁺ modified MgZnO ETLs. The photovoltaic and stability analysis of this improved photovoltaic performance is attributed to the suitable wide‐bandgap, low ETL/perovskite interface recombination, and interface stability by Mg²⁺ doping. Interestingly, the thermal stability analysis of the unencapsulated AI‐PSCs maintains >95% of initial PCE more than 400 h at 85 °C for MgZnO ETL, revealing the suitability against thermal degradation than conventional ZnO ETL.     

Identification of membrane associated drug targets in Borrelia burgdorferi ZS7- subtractive genomics approach

Lyme disease is an infectious disease caused by a spirochete Borrelia burgdorferi ZS7. This spirochete is most often spread by ticks. Single antibiotic therapy is sufficient for containment of the early stage progression of the disease but combinational therapy is more preferred in later stages. Research is in progress for the development of drugs against the pathogen, but till date no vaccines have been developed to effect the late stage infections. There is a rapid rise in the cases of antibiotic-resistant population which is more than 10% of the total infected individuals. In such condition vaccine becomes the sole alternative for prevention. Therefore effective treatment includes antibiotic combination and combination of antigenic surfaces (for vaccine preparation). Thus, a comprehensive list of drug targets unique to the microorganisms is often necessary. Availability of Borrelia burgdorferi ZS7 proteome has enabled insilico analysis of protein sequences for the identification of drug targets and vaccine targets. In this study, 272 essential proteins were identified out of which 42 proteins were unique to the microorganism. The study identified 15 membrane localized drug targets. Amongst these 15, molecular modeling and structure validation of the five membrane localized drug target proteins could only be achieved because of the low sequence identity of the remaining proteins with RCSB structures. These 3D structures can be further characterized by invitro and invivo studies for the development of novel vaccine epitopes and novel antibiotic therapy against Borrelia burgdorferi.     

Extraction and Sensitive Detection of Toxins A and B from the Human Pathogen Clostridium difficile in 40 Seconds Using Microwave-Accelerated Metal-Enhanced Fluorescence

Clostridium difficile is the primary cause of antibiotic associated diarrhea in humans and is a significant cause of morbidity and mortality. Thus the rapid and accurate identification of this pathogen in clinical samples, such as feces, is a key step in reducing the devastating impact of this disease. The bacterium produces two toxins, A and B, which are thought to be responsible for the majority of the pathology associated with the disease, although the relative contribution of each is currently a subject of debate. For this reason we have developed a rapid detection assay based on microwave-accelerated metal-enhanced fluorescence which is capable of detecting the presence of 10 bacteria in unprocessed human feces within 40 seconds. These promising results suggest that this prototype biosensor has the potential to be developed into a rapid, point of care, real time diagnostic assay for C. difficile.