The O-glycan is essential for the induction of protective antibodies against lethal infection by flagella A-bearing Pseudomonas aeruginosa.

To address the problem of increased antimicrobial resistance, we developed a glycoconjugate vaccine comprised of O-polysaccharides (OPS) of the four most prevalent serotypes of Klebsiella pneumoniae (KP) linked to recombinant flagellin types A and B (rFlaA and rFlaB) of Pseudomonas aeruginosa (PA). Flagellin is the major subunit of the flagellar filament. Flagella A and B, essential virulence factors for PA, are glycosylated with different glycans. We previously reported that while both rFlaA and rFlaB were highly immunogenic, only the rFlaB antisera reduced PA motility and protected mice from lethal PA infection in a mouse model of thermal injury. Since recombinant flagellin is not glycosylated, we examined the possibility that the glycan on native FlaA (nFlaA) might be critical to functional immune responses. We compared the ability of nFlaA to that of native, deglycosylated FlaA (dnFlaA) to induce functionally active antisera. O glycan was removed from nFlaA with trifluoromethanesulfonic acid. Despite the similar high-titered anti-FlaA antibody levels elicited by nFlaA, rFlaA, and dnFlaA, only the nFlaA antisera inhibited PA motility and protected mice following lethal intraperitoneal bacterial challenge. Both the protective efficacy and carrier protein function of nFlaA were retained when conjugated to KP O1 OPS. We conclude that unlike the case with FlaB O glycan, the FlaA glycan is an important epitope for the induction of functionally active anti-FlaA antibodies.

Mutations in linker histone genes HIST1H1 B, C, D, and E; OCT2 (POU2F2); IRF8; and ARID1A underlying the pathogenesis of follicular lymphoma.

Follicular lymphoma (FL) constitutes the second most common non-Hodgkin lymphoma in the western world. FL carries characteristic recurrent structural genomic aberrations. However, information regarding the coding genome in FL is still evolving. Here, we describe the results of massively parallel exome sequencing and single nucleotide polymorphism 6.0 array genomic profiling of 11 highly purified FL cases, and 1 transformed FL case and the validation of selected mutations in 102 FL cases. We report the identification of 15 novel recurrently mutated genes in FL. These include frequent mutations in the linker histone genes HIST1H1 B-E (27%) and mutations in OCT2 (also known as POU2F2; 8%), IRF8 (6%), and ARID1A (11%). A subset of the mutations in HIST1H1 B-E affected binding to DNMT3B, and mutations in HIST1H1 B-E and in EZH2 or ARID1A were largely mutually exclusive, implicating HIST1H1 B-E in epigenetic deregulation in FL. Mutations in OCT2 (POU2F2) affected its transcriptional and functional properties as measured through luciferase assays, the biological analysis of stably transduced cell lines, and global expression profiling. Finally, multiple novel mutated genes located within regions of acquired uniparental disomy in FL are identified. In aggregate, these data substantially broaden our understanding of the genomic pathogenesis of FL.

Characterizing human stem cell-derived sensory neurons at the single-cell level reveals their ion channel expression and utility in pain research.

The generation of human sensory neurons by directed differentiation of pluripotent stem cells opens new opportunities for investigating the biology of pain. The inability to generate this cell type has meant that up until now their study has been reliant on the use of rodent models. Here, we use a combination of population and single-cell techniques to perform a detailed molecular, electrophysiological, and pharmacological phenotyping of sensory neurons derived from human embryonic stem cells. We describe the evolution of cell populations over 6 weeks of directed differentiation; a process that results in the generation of a largely homogeneous population of neurons that are both molecularly and functionally comparable to human sensory neurons derived from mature dorsal root ganglia. This work opens the prospect of using pluripotent stem-cell-derived sensory neurons to study human neuronal physiology and as in vitro models for drug discovery in pain and sensory disorders.

Linear chromosome-generating system of Agrobacterium tumefaciens C58: protelomerase generates and protects hairpin ends.

Agrobacterium tumefaciens C58, the pathogenic bacteria that causes crown gall disease in plants, harbors one circular and one linear chromosome and two circular plasmids. The telomeres of its unusual linear chromosome are covalently closed hairpins. The circular and linear chromosomes co-segregate and are stably maintained in the organism. We have determined the sequence of the two ends of the linear chromosome thus completing the previously published genome sequence of A. tumefaciens C58. We found that the telomeres carry nearly identical 25-bp sequences at the hairpin ends that are related by dyad symmetry. We further showed that its Atu2523 gene encodes a protelomerase (resolvase) and that the purified enzyme can generate the linear chromosomal closed hairpin ends in a sequence-specific manner. Agrobacterium protelomerase, whose presence is apparently limited to biovar 1 strains, acts via a cleavage-and-religation mechanism by making a pair of transient staggered nicks invariably at 6-bp spacing as the reaction intermediate. The enzyme can be significantly shortened at both the N and C termini and still maintain its enzymatic activity. Although the full-length enzyme can uniquely bind to its product telomeres, the N-terminal truncations cannot. The target site can also be shortened from the native 50-bp inverted repeat to 26 bp; thus, the Agrobacterium hairpin-generating system represents the most compact activity of all hairpin linear chromosome- and plasmid-generating systems to date. The biochemical analyses of the protelomerase reactions further revealed that the tip of the hairpin telomere may be unusually polymorphically capable of accommodating any nucleotide.

Directing differentiation of human embryonic stem cells toward anterior neural ectoderm using small molecules.

Based on knowledge of early embryo development, where anterior neural ectoderm (ANE) development is regulated by native inhibitors of bone morphogenic protein (BMP) and Nodal/Activin signaling, most published protocols of human embryonic stem cell differentiation to ANE have demonstrated a crucial role for Smad signaling in neural induction. The drawbacks of such protocols include the use of an embryoid body culture step and use of polypeptide secreted factors that are both expensive and, when considering clinical applications, have significant challenges in terms of good manufacturing practices compliancy. The use of small molecules to direct differentiation of pluripotent stem cells toward a specified lineage represents a powerful approach to generate specific cell types for further understanding of biological function, for understanding disease processes, for use in drug discovery, and finally for use in regenerative medicine. We therefore aimed to find controlled and reproducible animal-component-free differentiation conditions that would use only small molecules. Here, we demonstrate that pluripotent stem cells can be reproducibly and efficiently differentiated to PAX6(+) (a marker of neuroectoderm) and OCT4(-) (a marker of pluripotent stem cells) cells with the use of potent small inhibitors of the BMP and Activin/Nodal pathways, and in animal-component-free conditions, replacing the frequently used Noggin and SB431542. We also show by transcript analysis, both at the population level and for the first time at the single-cell level, that differentiated cells express genes characteristic for the development of ANE, in particular for the development of the future forebrain.

Practical Management of the Venetoclax-Treated Patient in Chronic Lymphocytic Leukemia and Acute Myeloid Leukemia.

Venetoclax is a potent oral, highly selective small-molecule inhibitor of the antiapoptotic B-cell lymphoma 2 protein approved for chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma in treatment-naive patients (in combination with obinutuzumab) or for patients with relapsed/refractory CLL (in combination with rituximab). Venetoclax, in combination with azacitidine, decitabine, or low-dose cytarabine, is also approved in the United States for the treatment of newly diagnosed acute myeloid leukemia (AML) in adults who are ≥ 75 years or have comorbidities that preclude use of intensive induction chemotherapy. Clinical studies of patients with CLL or AML report both hematologic (e.g., neutropenia) and nonhematologic (e.g., gastrointestinal disorders and tumor lysis syndrome) adverse events associated with administration of venetoclax. It is therefore essential to provide information on the appropriate management of venetoclax-associated side effects. This article discusses the efficacy and safety of venetoclax administration and presents strategies specifically for the management of neutropenia and certain nonhematologic adverse events in patients receiving venetoclax for the treatment of AML and CLL.

Management of the airway in multitrauma.

PURPOSE OF REVIEW: The primary purpose of this article is to highlight the latest airway research in multitrauma. RECENT FINDINGS: Management of the airway in multitrauma patients is a critical resuscitation task. Prehospital airway management is difficult with a high risk of failure, complications, or both. In-hospital performed conventional oral intubation with manual in-line stabilization, cricoid pressure, and a backup plan for a surgical airway is still the most efficient and effective approach for early airway control in multitrauma patients. Selective utilization of airway maintenance, instead of ultimate airway control in the field, has been suggested as a primary prehospital strategy. Properties of videolaryngoscopes complement standard laryngoscopes. When compared with a Macintosh laryngoscope, the Airtraq and Airwayscope diminish cervical spine motion during elective orotracheal intubation. Penetrating neck injuries are the most frequent indication for awake intubation, whereas patients with maxillofacial injuries have the highest rate of initial surgical airway. SUMMARY: Risks and benefits of ultimate prehospital airway control is a controversial topic. Utilization of videolaryngoscopes in multitrauma remains open for research. Standardization of training requirements, equipment, and development of prehospital and in-hospital airway algorithms are needed to improve outcomes. Rational utilization of available airway devices, development of new devices, or both may help to promote this goal.

Financial difficulty in community-dwelling persons with lower limb loss is associated with reduced self-perceived health and wellbeing.

BACKGROUND: Socioeconomic status has been shown to be an important factor in the disparate prevalence and selected treatment of limb loss, but how personal financial difficulty affects patients' health outcomes is currently unclear. OBJECTIVE: Examining how presence and experience of personal financial difficulty affects perceived health and wellbeing in individuals with lower limb loss. STUDY DESIGN: Cross-sectional study. METHODS: A total of 90 participants (68 males, mean age 58.7 ± 16.7 years) were recruited from local physical therapy and prosthetic and orthotic clinics, rehabilitation hospitals, and a regional amputee patient support group. All participants were community-dwelling, non-military adults with amputation involving at least one major lower limb joint. Participants were interviewed, and each completed a survey that included basic demographic/medical information, self-reported health and wellbeing (Short-Form Health Survey, SF-36v2), and a question to determine their financial situation after limb loss. Multiple regression analyses were used to examine the effect of financial difficulty on the eight subscales of SF-36v2 while accounting for age, gender, and amputation level. RESULTS: Experiencing financial difficulty significantly and negatively affected Role-Physical and Role-Emotional subscale scores (p < 0.01 and p = 0.02, respectively). Individuals with financial difficulty scored approximately 60% lower in these two specific subscales. CONCLUSION: Experiencing financial difficulty is a significant predictor for diminished work or daily activity participation due to physical and emotional stresses. Clinicians and health policy makers need to understand how socioeconomic factors may prevent individuals with lower limb loss from achieving higher levels of functional recovery and community re-integration after amputation. CLINICAL RELEVANCE: Our findings showed that presence or experience of financial difficulty was significantly associated with diminished community re-integration in community-dwelling, non-military adults with lower limb loss. It affects both physical and emotional aspects of wellbeing. Clinicians should be aware how socioeconomic factors may affect social re-integration after amputation.

Initial stages of self-organization of silica-alumina gels in zeolite synthesis.

We report the effect of aluminum on the formation and structure of silica nanoparticles formed in basic solutions of small organic and inorganic cations using a combination of small-angle X-ray scattering, conductivity, pH, and 27Al NMR spectroscopy methods. At low silica concentrations, our observations agree with previous reports and show the formation of small oligomers ((HO)4-nAl(OSi(OH)3)n)- that can be modeled qualitatively using a simple aqueous speciation model. We also find that aluminum drastically reduces the concentration of silica at which nanoparticles are formed. Using organic cations, the particles are smaller than the ones observed in pure silica systems (1.5 nm vs approximately 3 nm diameter), but in the case of sodium cations, larger particles are detected ( approximately 10 nm in diameter). The data suggests that sodium cations are incorporated within the inorganic silica/alumina core, as opposed to organic cations that appear to be excluded from such clusters. Important insights are gained by making analogies to the behavior of mixed surfactant systems using regular solution theory and noting that the formation of Si-O-Al bonds (as measured by the DeltaGdegrees(rxn) of condensation) is much more favorable than the formation of Si-O-Si bonds.