Biocatalytic control of site-selectivity and chain length-selectivity in radical amino acid halogenases.

Biocatalytic C-H activation has the potential to merge enzymatic and synthetic strategies for bond formation. FeII/αKG-dependent halogenases are particularly distinguished for their ability both to control selective C-H activation as well as to direct group transfer of a bound anion along a reaction axis separate from oxygen rebound, enabling the development of new transformations. In this context, we elucidate the basis for the selectivity of enzymes that perform selective halogenation to yield 4-Cl-lysine (BesD), 5-Cl-lysine (HalB), and 4-Cl-ornithine (HalD), allowing us to probe how site-selectivity and chain length selectivity are achieved. We now report the crystal structure of the HalB and HalD, revealing the key role of the substrate-binding lid in positioning the substrate for C4 vs C5 chlorination and recognition of lysine vs ornithine. Targeted engineering of the substrate-binding lid further demonstrates that these selectivities can be altered or switched, showcasing the potential to develop halogenases for biocatalytic applications.

Engineering xylose induction in Vibrio natriegens for biomanufacturing applications.

Xylose is an abundant, inexpensive and readily available carbohydrate common in minimally processed feedstocks such as seaweed and algae. While a wide variety of marine microbes have evolved to utilize seaweed and algae, only a few currently have the requisite characteristics and genetic engineering tools necessary to entertain the use of these underutilized feedstocks. The rapidly growing Gram-negative halophilic bacterium Vibrio natriegens is one such chassis. In this study, we engineered and tested xylose induction in V. natriegens as a tool for scalable bioproduction applications. First, we created a sensing construct based on the xylose operon from Escherichia coli MG1665 and measured its activity using a fluorescent reporter and identified that cellular import plays a key role in induction strength and that expression required the XylR transcription factor. Next, we identified that select deletions of the promoter region enhance gene expression, limiting the effect of carbohydrate repression when xylose is used as an inducer in the presence of industrially relevant carbon sources. Lastly, we used the optimized constructs to produce the biopolymer melanin using seawater mimetic media. One of these formulations utilized a nori-based seaweed extract as an inducer and demonstrated melanin yields comparable to previously optimized methods using a more traditional and costly inducer. Together, the results demonstrate that engineering xylose induction in V. natriegens can provide an effective and lower cost option for timed biosynthesis in scalable biomanufacturing applications using renewable feedstocks.

Validation of LexisNexis Accurint in the Georgia Cancer Registry's Cancer Recurrence and Information Surveillance Program.

BACKGROUND: LexisNexis Accurint is a database of ~84 billion public records that includes an individual's location of residence. Its ability to track residences longitudinally has not been validated. This study used the Georgia Cancer Registry's (GCR's) Cancer Recurrence and Information Surveillance Program (CRISP) to validate the U.S. state of residence and to examine characteristics of patients not included or who had an inaccurate entry in LexisNexis. METHODS: The GCR is routinely linked to the National Death Index (NDI), providing information regarding the state of residence in which the patient died. We compared the state of residence reported in LexisNexis with the NDI gold standard state of residence at death. Multivariate logistic regression analyses estimated associations between demographic information and: (1) having a mismatch between LexisNexis and NDI and (2) being missed in LexisNexis. RESULTS: Of the 69,494 patients in the CRISP cohort, 65,890 (95%) were found in LexisNexis and 9,597 (14%) had died. Among a subset of patients who were deceased, the sensitivity of LexisNexis for identifying persons who left Georgia was 42% and the specificity was 89%. Minority groups were more likely to be missed in the LexisNexis database as well as to have discordance between LexisNexis and NDI state of residence at death. CONCLUSIONS: LexisNexis Accurint failed to identify the emigration of more than half of deceased CRISP patients who had left Georgia but correctly identified most who had remained. The validity of the state of residence is important for studies using LexisNexis as a tool for follow-up.

Accounting for red blood cell accessibility reveals distinct invasion strategies in Plasmodium falciparum strains.

The growth of the malaria parasite Plasmodium falciparum in human blood causes all the symptoms of malaria. To proliferate, non-motile parasites must have access to susceptible red blood cells, which they invade using pairs of parasite ligands and host receptors that define invasion pathways. Parasites can switch invasion pathways, and while this flexibility is thought to facilitate immune evasion, it may also reflect the heterogeneity of red blood cell surfaces within and between hosts. Host genetic background affects red blood cell structure, for example, and red blood cells also undergo dramatic changes in morphology and receptor density as they age. The in vivo consequences of both the accessibility of susceptible cells, and their heterogeneous susceptibility, remain unclear. Here, we measured invasion of laboratory strains of P. falciparum relying on distinct invasion pathways into red blood cells of different ages. We estimated invasion efficiency while accounting for red blood cell accessibility to parasites. This approach revealed different tradeoffs made by parasite strains between the fraction of cells they can invade and their invasion rate into them, and we distinguish "specialist" strains from "generalist" strains in this context. We developed a mathematical model to show that generalist strains would lead to higher peak parasitemias in vivo compared to specialist strains with similar overall proliferation rates. Thus, the ecology of red blood cells may play a key role in determining the rate of P. falciparum parasite proliferation and malaria virulence.

"Cure" Versus "Clinical Remission": The Impact of a Medication Description on the Willingness of People Living with HIV to Take a Medication.

Many people living with HIV (PLWHIV) state that they would be willing to take significant risks to be "cured" of the virus. However, how they interpret the word "cure" in this context is not clear. We used a randomized survey to examine whether PLWHIV had a different willingness to take a hypothetical HIV medication if it causes flu-like symptoms, but provides: (a) cure, (b) remission that was labeled "cure", or (c) remission. PLWHIV (n = 454) were more willing to take a medication that provided a "cure" versus a "remission" if the side effects lasted less than 1 year. PLWHIV were more willing to take a medication that provided a remission that was labeled "cure" versus a "remission" (p = 0.01) if the side effects lasted 2 weeks. Clinicians and researchers should be aware of the impact of the word "cure" and ensure that PLWHIV fully understand the possible outcomes of their treatment options.

YpeB dimerization may be required to stabilize SleB for effective germination of Bacillus anthracis spores.

BACKGROUND: Bacillus cells faced with unfavorable environmental conditions undergo an asymmetric division process ultimately leading to the formation of the bacterial spore. In some instances the spore serves as an infectious agent; such is the case with the spore of Bacillus anthracis and the disease anthrax. Spores are resistant to a variety of environment conditions including traditional decontamination techniques due to the formation of specialized cellular structures. One such structure, the spore cortex, is a thick layer of modified peptidoglycan that contributes to spore dormancy through maintenance of the dehydrated state of the spore core. During spore germination, degradation of the cortex is required to facilitate complete hydration of the core and a return to vegetative growth. Degradation of the cortex is accomplished through the action of germination-specific lytic enzymes. One of these enzymes, SleB, has been previously shown to require the presence of the YpeB protein for its stable incorporation and subsequent function in spores of B. anthracis. The focus of the present study is to identify protein interactions of YpeB through in vivo chemical cross-linking and two-hybrid analysis. RESULTS: Conserved residues within YpeB PepSY domains were altered to facilitate implementation of a site-specific chemical cross-linker, 4-Azidophenacyl bromide. Analyses of crosslinked-spore extracts suggests that YpeB exists as a dimer or larger multimer within the spore, potentially mediated through interactions of the C-terminal domains. Spores expressing stable truncated forms of YpeB were crosslinked and corresponding truncated dimers were detected. Further characterization of individual YpeB domains using bacterial two-hybrid analysis indicated a possible role for both N-and C-terminal domains in YpeB oligomerization. CONCLUSIONS: The YpeB protein likely exists as dimer or higher-order multimer in the dormant spore. Both the N- and C-terminal YpeB domains contribute to multimerization. SleB likely also exists as an oligomer, and SleB and YpeB may be found together within a protein complex. Disassembly of this complex during spore germination likely allows SleB to become active in spore cortex degradation. Further study of this protein complex may contribute to the development of methods to inhibit or stimulate germination, allowing more effective spore decontamination.

Efficient transposon mutagenesis mediated by an IPTG-controlled conditional suicide plasmid.

BACKGROUND: Transposon mutagenesis is highly valuable for bacterial genetic and genomic studies. The transposons are usually delivered into host cells through conjugation or electroporation of a suicide plasmid. However, many bacterial species cannot be efficiently conjugated or transformed for transposon saturation mutagenesis. For this reason, temperature-sensitive (ts) plasmids have also been developed for transposon mutagenesis, but prolonged incubation at high temperatures to induce ts plasmid loss can be harmful to the hosts and lead to enrichment of mutants with adaptive genetic changes. In addition, the ts phenotype of a plasmid is often strain- or species-specific, as it may become non-ts or suicidal in different bacterial species. RESULTS: We have engineered several conditional suicide plasmids that have a broad host range and whose loss is IPTG-controlled. One construct, which has the highest stability in the absence of IPTG induction, was then used as a curable vector to deliver hyperactive miniTn5 transposons for insertional mutagenesis. Our analyses show that these new tools can be used for efficient and regulatable transposon mutagenesis in Escherichia coli, Acinetobacter baylyi and Pseudomonas aeruginosa. In P. aeruginosa PAO1, we have used this method to generate a Tn5 insertion library with an estimated diversity of ~ 108, which is ~ 2 logs larger than the best transposon insertional library of PAO1 and related Pseudomonas strains previously reported. CONCLUSION: We have developed a number of IPTG-controlled conditional suicide plasmids. By exploiting one of them for transposon delivery, a highly efficient and broadly useful mutagenesis system has been developed. As the assay condition is mild, we believe that our methodology will have broad applications in microbiology research.

Directed evolution of the Escherichia coli cAMP receptor protein at the cAMP pocket.

The Escherichia coli cAMP receptor protein (CRP) requires cAMP binding to undergo a conformational change for DNA binding and transcriptional regulation. Two CRP residues, Thr(127) and Ser(128), are known to play important roles in cAMP binding through hydrogen bonding and in the cAMP-induced conformational change, but the connection between the two is not completely clear. Here, we simultaneously randomized the codons for these two residues and selected CRP mutants displaying high CRP activity in a cAMP-producing E. coli. Many different CRP mutants satisfied the screening condition for high CRP activity, including those that cannot form any hydrogen bonds with the incoming cAMP at the two positions. In vitro DNA-binding analysis confirmed that these selected CRP mutants indeed display high CRP activity in response to cAMP. These results indicate that the hydrogen bonding ability of the Thr(127) and Ser(128) residues is not critical for the cAMP-induced CRP activation. However, the hydrogen bonding ability of Thr(127) and Ser(128) was found to be important in attaining high cAMP affinity. Computational analysis revealed that most natural cAMP-sensing CRP homologs have Thr/Ser, Thr/Thr, or Thr/Asn at positions 127 and 128. All of these pairs are excellent hydrogen bonding partners and they do not elevate CRP activity in the absence of cAMP. Taken together, our analyses suggest that CRP evolved to have hydrogen bonding residues at the cAMP pocket residues 127 and 128 for performing dual functions: preserving high cAMP affinity and keeping CRP inactive in the absence of cAMP.

Genetic markers of repolarization and arrhythmic events after acute coronary syndromes.

BACKGROUND: There is a genetic contribution to the risk of ventricular arrhythmias in survivors of acute coronary syndromes (ACS). We wished to explore the role of 33 candidate single nucleotide polymorphisms (SNPs) in prolonged repolarization and sudden death in patients surviving ACS. METHODS: A total of 2,139 patients (1680 white ethnicity) surviving an admission for ACS were enrolled in the prospective Coronary Disease Cohort Study. Extensive clinical, echocardiographic, and neurohormonal data were collected for 12 months, and clinical events were recorded for a median of 5 years. Each SNP was assessed for association with sudden cardiac death (SCD)/cardiac arrest (CA) and prolonged repolarization at 3 time-points: index admission, 1 month, and 12 months postdischarge. RESULTS: One hundred six SCD/CA events occurred during follow-up (6.3%). Three SNPs from 3 genes (rs17779747 [KCNJ2], rs876188 [C14orf64], rs3864180 [GPC5]) were significantly associated with SCD/CA in multivariable models (after correction for multiple testing); the minor allele of rs17779747 with a decreased risk (hazard ratio [HR] 0.68 per copy of the minor allele, 95% CI 0.50-0.92, P = .012), and rs876188 and rs386418 with an increased risk (HR 1.52 [95% CI 1.10-2.09, P = .011] and HR 1.34 [95% CI 1.04-1.82, P = .023], respectively). At 12 months postdischarge, rs10494366 and rs12143842 (NOS1AP) were significant predictors of prolonged repolarization (HR 1.32 [95% CI 1.04-1.67, P = .022] and HR 1.30 [95% CI 1.01-1.66, P = .038], respectively), but not at earlier time-points. CONCLUSION: Three SNPs were associated with SCD/CA. Repolarization time was associated with variation in the NOS1AP gene. This study demonstrates a possible role for SNPs in risk stratification for arrhythmic events after ACS.

Adeno-Associated Virus Type 9-Mediated Gene Therapy of Choline Acetyltransferase-Deficient Mice.

The enzyme choline acetyltransferase (ChAT) synthesizes acetylcholine from acetyl-CoA and choline at the neuromuscular junction and at the nerve terminals of cholinergic neurons. Mutations in the ChAT gene (CHAT) result in a presynaptic congenital myasthenic syndrome (CMS) that often associates with life-threatening episodes of apnea. Knockout mice for Chat (Chat-/-) die at birth. To circumvent the lethality of this model, we crossed mutant mice possessing loxP sites flanking Chat exons 4 and 5 with mice that expressed Cre-ERT2. Injection of tamoxifen (Tx) at postnatal (P) day 11 in these mice induced downregulation of Chat, autonomic failure, weakness, and death. However, a proportion of Chatflox/flox-Cre-ERT2 mice receiving at birth an intracerebroventricular injection of 2 × 1013 vg/kg adeno-associated virus type 9 (AAV9) carrying human CHAT (AAV9-CHAT) survived a subsequent Tx injection and lived to adulthood without showing signs of weakness. Likewise, injection of AA9-CHAT by intracisternal injection at P28 after the onset of weakness also resulted in survival to adulthood. The expression of Chat in spinal motor neurons of Chatflox/flox-Cre-ERT2 mice injected with Tx was markedly reduced, but AAV-injected mice showed a robust recovery of ChAT expression, which was mainly translated by the human CHAT RNA. The biodistribution of the viral genome was widespread but maximal in the spinal cord and brain of AAV-injected mice. No significant histopathological changes were observed in the brain, liver, and heart of AAV-injected mice after 1 year follow-up. Thus, AAV9-mediated gene therapy may provide an effective and safe treatment for patients severely affected with CHAT-CMS.

Impact of age, phenotype and cardio-renal function on plasma C-type and B-type natriuretic peptide forms in an adult population.

CONTEXT: In contrast to the cardiac hormones, atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP), variations in plasma concentrations of C-type natriuretic peptide (CNP) in healthy adults are ill-defined, limiting their clinical application. OBJECTIVE: Our objective was to define the effect of age, phenotype (gender, height, BMI), and cardiac and renal function on plasma CNPs in an adults population without renal or cardiovascular disease. DESIGN AND SETTING: This was a prospective cross-sectional observational study of adult volunteers, aged 21-80 years, randomly selected from the electoral roll. SUBJECTS AND METHODS: Plasma CNP and its associated aminoterminal propeptide (NTproCNP) were measured in 258 subjects and related to age, gender, height and plasma creatinine. Subgroup analyses seeking associations with cardiac function (plasma BNP and NTproBNP) and bone turnover bone-specific alkaline phosphatase (bALP) were also determined. RESULTS: Plasma concentrations of CNPs in men continued to decline from adolescent values to reach a nadir in the 5th decade after which values increased. Similar but less marked changes occurred in women. In both sexes, NTproCNP was inversely and independently correlated with height. In contrast to B-type natriuretic peptides (BNPs), NTproCNP was higher in men, significantly related to creatinine and positively related to bALP. CONCLUSIONS: Gender- and age-specific changes affect CNPs in adults. Inverse associations of NTproCNP with adult height, positive correlation with creatinine - and in contrast to CNP - no association with BNP are further unique findings distinguishing NTproCNP, which need to be considered in future studies.

Hyperuricaemia and gout in New Zealand rural and urban Maori and non-Maori communities.

BACKGROUND: There are few current data on the prevalence of hyperuricaemia and gout in New Zealand, particularly among the indigenous Maori population. AIMS: To determine the prevalence of gout and hyperuricaemia in rural and urban Maori and non-Maori community samples and describe the treatment and comorbidities of participants with gout. METHODS: Participants aged 20-64 years were recruited by random selection from the electoral roll. Maori samples were selected from among those identified as being of Maori descent on the roll and who self-identified as being of Maori ethnicity at interview. Personal medical history, blood pressure, anthropometrics, fasting lipids, glucose, HbA1c and urate were recorded. RESULTS: There were 751 participants. Mean serum urate (SU) was 0.30 mmol/L (0.06-0.69 mmol/L). Maori had a significantly higher prevalence of hyperuricaemia (SU > 0.40 mmol/L) compared with non-Maori (17.0% vs 7.5%, P = 0.0003). A total of 57 participants had a history of gout, with a higher prevalence in Maori compared with non-Maori (10.3% vs 2.3%, P < 0.0001). Of the participants, 18/57 (31.6%) with gout were receiving urate-lowering therapy, but in 38.9%, SU was >0.36 mmol/L. Participants with gout were more likely to have metabolic syndrome, diabetes, cardiac disease or hypertension. CONCLUSIONS: Gout and hyperuricaemia were more prevalent in Maori, and participants with gout were more likely to have comorbidities. There was not a higher overall adjusted cardiovascular disease risk in Maori participants with gout. Despite the high prevalence of gout, management remains suboptimal.

Expanding the substrate scope of pyrrolysyl-transfer RNA synthetase enzymes to include non-α-amino acids in vitro and in vivo.

The absence of orthogonal aminoacyl-transfer RNA (tRNA) synthetases that accept non-L-α-amino acids is a primary bottleneck hindering the in vivo translation of sequence-defined hetero-oligomers and biomaterials. Here we report that pyrrolysyl-tRNA synthetase (PylRS) and certain PylRS variants accept α-hydroxy, α-thio and N-formyl-L-α-amino acids, as well as α-carboxy acid monomers that are precursors to polyketide natural products. These monomers are accommodated and accepted by the translation apparatus in vitro; those with reactive nucleophiles are incorporated into proteins in vivo. High-resolution structural analysis of the complex formed between one PylRS enzyme and a m-substituted 2-benzylmalonic acid derivative revealed an active site that discriminates prochiral carboxylates and accommodates the large size and distinct electrostatics of an α-carboxy substituent. This work emphasizes the potential of PylRS-derived enzymes for acylating tRNA with monomers whose α-substituent diverges substantially from the α-amine of proteinogenic amino acids. These enzymes or derivatives thereof could synergize with natural or evolved ribosomes and/or translation factors to generate diverse sequence-defined non-protein heteropolymers.

Free Ig light chains interact with sphingomyelin and are found on the surface of myeloma plasma cells in an aggregated form.

Free κ L chains (FκLCs) are expressed on the surface of myeloma cells and are being assessed as a therapeutic target for the treatment of multiple myeloma. Despite its clinical potential, the mechanism by which FκLCs interact with membranes remains unresolved. In this study, we show that FκLCs associate with sphingomyelin on the plasma membrane of myeloma cells. Moreover, membrane-bound FκLCs are aggregated, suggesting that aggregation is required for intercalation with membranes. Finally, we propose a model where the binding of FκLCs with sphingomyelin on secretory vesicle membranes is stabilized by self-aggregation, with aggregated FκLCs exposed on the plasma membrane after exocytosis. Although it is well known that protein aggregates bind membranes, this is only the second example of an aggregate being found on the surface of cells that also secrete the protein in its native form. We postulate that many other aggregation-prone proteins may associate with cell membranes by similar mechanisms.

Methylsulfonylmethane and Sesame Seed Oil Improve Dyslipidemia and Modulate Polyunsaturated Fatty Acid Metabolism in Two Mouse Models of Diabetes.

The objective of this study was to identify alterations in lipids and polyunsaturated fatty acid (PUFA) metabolism in both the streptozotocin (STZ)-induced type 1 diabetic (T1D) mouse and the mutant db/db type 2 diabetic (T2D) mouse to establish a biological signature for the evaluation of natural products with purported lipid-altering activity. Eight-week-old male C57BL/6J mice were randomized to nondiabetic group or STZ-induced diabetic groups (n = 10/group). STZ-induced diabetic mice and 6-week-old male db/db mice (n = 10/group) were randomized to the following groups: (1) diabetic control, no treatment, (2) methylsulfonylmethane (MSM) treatment, (3) sesame seed oil (SSO) treatment, and (4) MSM+SSO combination treatment. Clinical parameters measured included weights, blood glucose, serum lipid panels, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) detection of free fatty acids in serum, liver, brain, and eyes. Blood glucose significantly decreased after 4 weeks of MSM treatment in T1D mice. Serum PUFA levels were significantly reduced in T2D mice compared with control mice. In contrast, treatment with SSO reversed this effect in T2D mice, exhibiting serum PUFA levels comparable to control mice. Serum triglycerides were significantly increased in both diabetic models compared to nondiabetic control, mimicking diabetes in people. High-density lipoprotein (HDL) was significantly increased in T1D receiving MSM+SSO and all T2D treatment groups. A corresponding significant decrease in non-HDL cholesterol was seen in T2D mice in all treatment groups. MSM+SSO treatment's effects on HDL and non-HDL cholesterol and PUFA metabolism could lead to improved clinical outcomes in diabetics by improving the lipid profile.

Suggestive evidence for Darwinian Selection against asparagine-linked glycans of Plasmodium falciparum and Toxoplasma gondii.

We are interested in asparagine-linked glycans (N-glycans) of Plasmodium falciparum and Toxoplasma gondii, because their N-glycan structures have been controversial and because we hypothesize that there might be selection against N-glycans in nucleus-encoded proteins that must pass through the endoplasmic reticulum (ER) prior to threading into the apicoplast. In support of our hypothesis, we observed the following. First, in protists with apicoplasts, there is extensive secondary loss of Alg enzymes that make lipid-linked precursors to N-glycans. Theileria makes no N-glycans, and Plasmodium makes a severely truncated N-glycan precursor composed of one or two GlcNAc residues. Second, secreted proteins of Toxoplasma, which uses its own 10-sugar precursor (Glc(3)Man(5)GlcNAc(2)) and the host 14-sugar precursor (Glc(3)Man(9)GlcNAc(2)) to make N-glycans, have very few sites for N glycosylation, and there is additional selection against N-glycan sites in its apicoplast-targeted proteins. Third, while the GlcNAc-binding Griffonia simplicifolia lectin II labels ER, rhoptries, and surface of plasmodia, there is no apicoplast labeling. Similarly, the antiretroviral lectin cyanovirin-N, which binds to N-glycans of Toxoplasma, labels ER and rhoptries, but there is no apicoplast labeling. We conclude that possible selection against N-glycans in protists with apicoplasts occurs by eliminating N-glycans (Theileria), reducing their length (Plasmodium), or reducing the number of N-glycan sites (Toxoplasma). In addition, occupation of N-glycan sites is markedly reduced in apicoplast proteins versus some secretory proteins in both Plasmodium and Toxoplasma.

Gel Polymer Electrolytes Based on Cross-Linked Poly(ethylene glycol) Diacrylate for Calcium-Ion Conduction.

Calcium batteries are promising alternatives to lithium batteries owing to their high energy density, comparable reduction potential, and mineral abundance. However, to meet practical demands in high-performance applications, suitable electrolytes must be developed. Here, we report the synthesis and characterization of polymer gel electrolytes for calcium-ion conduction prepared by the photo-cross-linking of poly(ethylene glycol) diacrylate (PEGDA) in the presence of solutions of calcium salts in a mixture of ethylene carbonate (EC) and propylene carbonate (PC) solvents. The results show room-temperature conductivity between 10-5 and 10-4 S/cm, electrochemical stability windows of ∼3.8 V, full dissociation of the salt, and minimal coordination with the PEGDA backbone. Cycling in symmetric Ca metal cells proceeds but with increasing overpotentials, which can be attributed to interfacial impedance between the electrolyte and calcium surface, which inhibits charge transfer. Calcium may still be plated and stripped yielding high-purity deposits and no indication of significant electrolyte breakdown, indicating that high overpotentials are associated with an electrically insulating, yet ion-permeable solid electrolyte interface (SEI). This work provides a contribution to the study and understanding of polymer gel materials toward their improvement and application as electrolytes for calcium batteries.

Cooperativity between Plasmodium falciparum adhesive proteins for invasion into erythrocytes.

Plasmodium falciparum is the most virulent of the Plasmodium species infective to humans. Different P. falciparum strains vary in their dependence on erythrocyte receptors for invasion and their ability to switch in their utilization of different receptor repertoires. Members of the reticulocyte-binding protein-like (RBL) family of invasion ligands are postulated to play a central role in defining ligand-receptor interactions, known as invasion pathways. Here we report the targeted gene disruption of PfRh2b and PfRh2a in W2mef, a parasite strain that is heavily dependent on sialic-acid receptors for invasion, and show that the PfRh2b ligand is functional in this parasite background. Like the parental line, parasites lacking either PfRh2a or PfR2b can switch to a sialic acid-independent invasion pathway. However, both of the switched lines exhibit a reduced efficiency for invasion into sialic acid-depleted cells, suggesting a role for both PfRh2b and PfRh2a in invasion via sialic acid-independent receptors. We also find a strong selective pressure for the reconstitution of PfRh2b expression at the expense of PfRh2a. Our results reveal the importance of genetic background in ligand-receptor usage by P. falciparum parasites, and suggest that the co-ordinate expression of PfRh2a, PfRh2b together mediate efficient sialic acid-independent erythrocyte invasion.

Identification of L-Valine-initiated-germination-active genes in Bacillus subtilis using Tn-seq.

Bacterial endospores can survive harsh environmental conditions and long-term dormancy in the absence of nutrients, but can rapidly germinate under favorable conditions. In the present study, we employed transposon sequencing (Tn-seq) to identify genes with previously uncharacterized roles in spore germination. Identified genes that encoded spore inner membrane proteins were chosen for study of defined mutants, which exhibited delayed germination in several assays in response to varying germinants. Significantly slowed release of DPA indicated that mutants were affected in Stage I of germination. Several mutants exhibited phenotypic traits consistent with failure of a GerA germinant receptor-mediated response, while others appeared to have a more general loss of response to varied germinants. Use of a gerA-lacZ transcriptional fusion and quantitative western blotting of GerAC allowed mutants to be classified based upon normal or decreased gerA transcription and normal or reduced GerA accumulation. Fourteen genes were identified to have newly described roles within Bacillus spore germination. A more complete understanding of this process can contribute to the development of better spore decontamination procedures.

Nanotechnology in regenerative ophthalmology.

In recent years, regenerative medicine is gaining momentum and is giving hopes for restoring function of diseased, damaged, and aged tissues and organs and nanotechnology is serving as a catalyst. In the ophthalmology field, various types of allogenic and autologous stem cells have been investigated to treat some ocular diseases due to age-related macular degeneration, glaucoma, retinitis pigmentosa, diabetic retinopathy, and corneal and lens traumas. Nanomaterials have been utilized directly as nanoscaffolds for these stem cells to promote their adhesion, proliferation and differentiation or indirectly as vectors for various genes, tissue growth factors, cytokines and immunosuppressants to facilitate cell reprogramming or ocular tissue regeneration. In this review, we reviewed various nanomaterials used for retina, cornea, and lens regenerations, and discussed the current status and future perspectives of nanotechnology in tracking cells in the eye and personalized regenerative ophthalmology. The purpose of this review is to provide comprehensive and timely insights on the emerging field of nanotechnology for ocular tissue engineering and regeneration.