Genomic analysis of Kazachstania aerobia and Kazachstania servazzii reveals duplication of genes related to acetate ester production.

Kazachstania aerobia and Kazachstania servazzii can affect wine aroma by increasing acetate ester concentrations, most remarkably phenylethyl acetate and isoamyl acetate. The genetic basis of this is unknown, there being little to no sequence data available on the genome architecture. We report for the first time the near-complete genome sequence of the two species using long-read (PacBio) sequencing (K. aerobia 20 contigs, one scaffold; and K. servazzii 22 contigs, one scaffold). The annotated genomes of K. aerobia (12.5 Mb) and K. servazzii (12.3 Mb) were compared to Saccharomyces cerevisiae genomes (laboratory strain S288C and wine strain EC1118). Whilst a comparison of the two Kazachstania spp. genomes revealed few differences between them, divergence was evident in relation to the genes involved in ester biosynthesis, for which gene duplications or absences were apparent. The annotations of these genomes are valuable resources for future research into the evolutionary biology of Kazachstania and other yeast species (comparative genomics) as well as understanding the metabolic processes associated with alcoholic fermentation and the production of secondary 'aromatic' metabolites (transcriptomics, proteomics and metabolomics).

Modern yeast development: finding the balance between tradition and innovation in contemporary winemaking.

A key driver of quality in wines is the microbial population that undertakes fermentation of grape must. Winemakers can utilise both indigenous and purposefully inoculated yeasts to undertake alcoholic fermentation, imparting wines with aromas, flavours and palate structure and in many cases contributing to complexity and uniqueness. Importantly, having a toolbox of microbes helps winemakers make best use of the grapes they are presented with, and tackle fermentation difficulties with flexibility and efficiency. Each year the number of strains available commercially expands and more recently, includes strains of non-Saccharomyces, strains that have been improved using both classical and modern yeast technology and mixed cultures. Here we review what is available commercially, and what may be in the future, by exploring recent advances in fermentation relevant strain improvement technologies. We also report on the current use of microbes in the Australian wine industry, as reported by winemakers, as well as regulations around, and sentiment about the potential use of genetically modified organisms in the future.

Characterization of polysulfides in Saccharomyces cerevisiae cells and finished wine from a cysteine-supplemented model grape medium.

Polysulfide degradation in wine can result in hydrogen sulfide (H2S) release, imparting a rotten-egg smell that is detrimental to wine quality. Although the presence of wine polysulfides has been demonstrated, their biogenesis remains unclear. This study investigated the role of Saccharomyces cerevisiae in polysulfide formation during fermentation, with and without 5 mM cysteine supplementation as an H2S source. Using an established liquid chromatography-tandem mass spectrometry method, monobromobimane derivatives of hydropolysulfides, including CysSSSH, CysSSSSH and GSSSSH, and two oxidized polysulfides, GSSG and GSSSSG, were detected in yeast cells at the end of fermentation in a grape juice-like medium. Polysulfide production by four S. cerevisiae single deletion mutants (BY4743 Δcys3, Δcys4, Δmet17 and Δtum1) showed no significant differences compared to BY4743, suggesting that uncharacterized pathways maintain cellular polysulfide homeostasis. Five mM cysteine addition increased the formation of shorter sulfur chain species, including GSS-bimane and GSSG, but did not elevate levels of longer sulfur chain species. Additionally, polysulfides with even numbers of sulfur atoms tended to predominate in cellular lysates. Oxidized polysulfides and longer chain hydropolysulfides were not detected in finished wines. This evidence suggests that these polysulfides are unstable in wine-like environments or not transported extracellularly. Collectively, our data illustrate the complexity of yeast polysulfide metabolism under fermentation conditions.

Case presentation of 8-year follow up of recurrent malignant duodenal Insulinoma and lymph node metastases and literature review of malignant Insulinoma management.

BACKGROUND: Insulinoma is an uncommon insulin-secreting neuroendocrine tumor that presents with severe recurrent hypoglycemia. Although cases of extrapancreatic insulinomas have been reported, the majority of insulinomas occur in the pancreas. The number of reported cases of ectopic insulinomas with follow-up assessments is limited and they do not report disease recurrence. The current report presents the first documented case of recurrent extrapancreatic insulinoma with 8 years of follow-up, provides relevant literature review, and proposes surveillance and treatment strategies. CASE PRESENTATION: We describe an insulinoma localized in the duodenal wall of a 36-year-old female who presented in 2013 with weight gain and Whipple's triad and was successfully managed with duodenotomy and enucleation. She presented again in 2017 with recurrent Whipple's triad and was found to have metastatic disease localized exclusively to peripancreatic lymph nodes. Primary pancreatic insulinoma was not evident and her hypoglycemia resolved following lymph node dissection. Eight years after initial presentation continuous glucose monitoring (CGM) showed a trend for euglycemia, and PET-CT Gallium 68 DOTATATE scan evaluation indicated absence of recurrent disease. CONCLUSION: Insulinomas are rare clinical entities and extrapancreatic insulinomas are particularly uncommon. Follow-up evaluation and treatment strategies for ectopic insulinoma recurrence presents a significant clinical challenge as the condition has hitherto remained undescribed in the literature. Available evidence in the literature indicates that lymph node metastases of intrapancreatic insulinomas likely do not change prognosis. Given the absence of long-term data informing the management and monitoring of patients with extrapancreatic insulinoma, we suggest patient education for hypoglycemic symptoms, monitoring for hypoglycemia with CGM, annual imaging, and a discussion with patients regarding treatment with octreotide or alternative somatostatin receptor analog therapies.

Improving Patient Safety and Quality in Physical Medicine and Rehabilitation Through Participation in the American Board of Physical Medicine and Rehabilitation Continuing Certification Program.

ABSTRACT: The American Board of Medical Specialties Continuing Certification Program's Improvement in Medical Practice Standard requires physicians to participate in practice improvement activities. Despite this universal requirement, there has been no assessment of this requirement or its potential impact on patient care. Because of its continuing certification oversight structure, the American Board of Physical Medicine and Rehabilitation is in a unique position to provide this assessment. Review of quality improvement projects submitted to the American Board of Physical Medicine and Rehabilitation for continuing certification compliance revealed that most diplomates (70.1%) used available topic-specific options. These projects are designed to be directive and easy to use for physicians with limited quality improvement experience. Examples of topic-directed project potential impact on patient care include preventing wrong-site injections through implementing a preprocedure timeout or decreasing opioid prescribing risk through implementation of an opioid risk assessment tool. Thirty percent of submissions described improvement efforts in other areas of practice. These projects were directed toward areas of patient care including safety, communication/education, satisfaction, processes, and outcomes. This study demonstrates the efforts of physiatrists to improve care and the potential impact of these efforts on patient care and safety through participation in continuing certification.

Directed evolution as an approach to increase fructose utilization in synthetic grape juice by wine yeast AWRI 796.

In winemaking, slow or stuck alcoholic fermentation can impact processing efficiency and wine quality. Residual fructose in the later stages of fermentation can leave the wine 'out of specification' unless removed, which requires reinoculation or use of a more fructophilic yeast. As such, robust, fermentation efficient strains are still highly desirable to reduce this risk. We report on a combined EMS mutagenesis and Directed Evolution (DE) approach as a 'proof of concept' to improve fructose utilization and decrease fermentation duration. One evolved isolate, Tee 9, was evaluated against the parent, AWRI 796 in defined medium (CDGJM) and Semillon juice. Interestingly, Tee 9 exhibited improved fermentation in CDGJM at several nitrogen contents, but not in juice. Genomic comparison between AWRI 796 and Tee 9 identified 371 mutations, but no chromosomal copy number variation. A total of 95 noncoding and 276 coding mutations were identified in 297 genes (180 of which encode proteins with one or more substitutions). Whilst introduction of two of these, Gid7 (E726K) or Fba1 (G135S), into AWRI 796 did not lead to the fermentation improvement seen in Tee 9, similar allelic swaps with the other mutations are needed to understand Tee 9's adaption to CDGJM. Furthermore, the 378 isolates, potentially mutagenized but with the same genetic background, are likely a useful resource for future phenotyping and genome-wide association studies.

Influence of Kazachstania spp. on the chemical and sensory profile of red wines.

We report the fermentative traits of two Kazachstania species (K. aerobia and K. servazzii) in non-sterile red wine and the resulting chemical and sensory properties. This builds on our previous work which revealed that Kazachstania spp. increased acetate esters in sterilised white wine. In this study Kazachstania spp. were initially evaluated in laboratory-scale fermentations (500 mL) in Merlot must to assess whether similar increases in chemical/volatile compounds would occur. The impact of malolactic fermentation (MLF) by Oenococcus oeni (VP41) on aroma composition was considered and found to reduce ester profiles in Merlot wines. The sensory implications of sequential inoculation with Kazachstania spp., followed by Saccharomyces cerevisiae, were then evaluated in small-lot fermentations (7 kg) of Shiraz must. Fungal diversity was monitored during early fermentation stages and was influenced by the early implantation of Kazachstania spp., followed by the dominance of S. cerevisiae. The effect of MLF in Shiraz wines was inconclusive due to high ethanol levels providing an inhospitable environment for lactic acid bacteria. When compared to S. cerevisiae alone, Kazachstania spp. significantly increased acetate esters, particularly phenylethyl acetate and isoamyl acetate, in both Merlot and Shiraz. The Shiraz wines fermented with Kazachstania spp. had higher jammy and red fruit aroma/flavour compared to S. cerevisiae (monoculture) wines. No influence was observed on colour one-year post-bottling. Results from this study show the contribution of Kazachstania spp. to the aroma profile of red wines and demonstrate their potential as starter cultures for improving the aromatic complexity of wines.

Exploring the diversity of bacteriophage specific to Oenococcus oeni and Lactobacillus spp and their role in wine production.

The widespread existence of bacteriophage has been of great interest to the biological research community and ongoing investigations continue to explore their diversity and role. They have also attracted attention and in-depth research in connection to fermented food processing, in particular from the dairy and wine industries. Bacteriophage, mostly oenophage, may in fact be a 'double edged sword' for winemakers: whilst they have been implicated as a causal agent of difficulties with malolactic fermentation (although not proven), they are also beginning to be considered as alternatives to using sulphur dioxide to prevent wine spoilage. Investigation and characterisation of oenophage of Oenococcus oeni, the main species used in winemaking, are still limited compared to lactococcal bacteriophage of Lactococcus lactis and Lactiplantibacillus plantarum (formally Lactobacillus plantarum), the drivers of most fermented dairy products. Interestingly, these strains are also being used or considered for use in winemaking. In this review, the genetic diversity and life cycle of phage, together with the debate on the consequent impact of phage predation in wine, and potential control strategies are discussed. KEY POINTS: • Bacteriophage detected in wine are diverse. • Many lysogenic bacteriophage are found in wine bacteria. • Phage impact on winemaking can depend on the stage of the winemaking process. • Bacteriophage as potential antimicrobial agents against spoilage organisms.

Disruption of ECM33 in diploid wine yeast EC1118: cell morphology and aggregation and their influence on fermentation performance.

When investigating yeast gene function in relation to fermentation, many screens rely on haploid yeast derivatives. This, however, is not representative of industrial strains, which are typically diploid. One such example is the disruption of ECM33, which was associated with improved fermentation in the haploid wine yeast C911D, but remains uncharacterised in a diploid industrial strain background. We report on the homozygous disruption of ECM33 in Lalvin EC1118 using CRISPR/Cas9. EC1118 ecm33 resulted in a reduction of fermentation duration in a defined medium with limiting and sufficient nitrogen (-20% and -13%, respectively) when shaken. Increased cell size and aggregation, a phenotype previously unidentified in ecm33∆ as haploid yeast tend to aggregate, was also observed. This phenotype led to premature settling thereby the yeast behaving similarly to EC1118 in wine-like semi-static fermentations in a chemically defined medium. Further assessment in semi-static Riesling and Chardonnay fermentations inoculated based on cell number or biomass resulted in no significant difference or significantly slower fermentation duration in comparison the EC1118, nullifying the benefits of this mutation unless agitation is applied. This study draws attention to phenotypes being condition-dependent, highlighting the need to characterise and verify fermentation efficiency mutations in industrial yeast.

"The talking bit of medicine, that's the most important bit": doctors and Aboriginal interpreters collaborate to transform culturally competent hospital care.

BACKGROUND: In hospitals globally, patient centred communication is difficult to practice, and interpreters are underused. Low uptake of interpreters is commonly attributed to limited interpreter availability, time constraints and that interpreter-medicated communication in healthcare is an aberration. In Australia's Northern Territory at Royal Darwin Hospital, it is estimated around 50% of Aboriginal patients would benefit from an interpreter, yet approximately 17% get access. Recognising this contributes to a culturally unsafe system, Royal Darwin Hospital and the NT Aboriginal Interpreter Service embedded interpreters in a renal team during medical ward rounds for 4 weeks in 2019. This paper explores the attitudinal and behavioural changes that occurred amongst non-Indigenous doctors and Aboriginal language interpreters during the pilot. METHODS: This pilot was part of a larger Participatory Action Research study examining strategies to achieve culturally safe communication at Royal Darwin Hospital. Two Yolŋu and two Tiwi language interpreters were embedded in a team of renal doctors. Data sources included interviews with doctors, interpreters, and an interpreter trainer; reflective journals by doctors; and researcher field notes. Inductive thematic analysis, guided by critical theory, was conducted. RESULTS: Before the pilot, frustrated doctors unable to communicate effectively with Aboriginal language speaking patients acknowledged their personal limitations and criticised hospital systems that prioritized perceived efficiency over interpreter access. During the pilot, knowledge of Aboriginal cultures improved and doctors adapted their work routines including lengthening the duration of bed side consults. Furthermore, attitudes towards culturally safe communication in the hospital changed: doctors recognised the limitations of clinically focussed communication and began prioritising patient needs and interpreters who previously felt unwelcome within the hospital reported feeling valued as skilled professionals. Despite these benefits, resistance to interpreter use remained amongst some members of the multi-disciplinary team. CONCLUSIONS: Embedding Aboriginal interpreters in a hospital renal team which services predominantly Aboriginal peoples resulted in the delivery of culturally competent care. By working with interpreters, non-Indigenous doctors were prompted to reflect on their attitudes which deepened their critical consciousness resulting in behaviour change. Scale up of learnings from this pilot to broader implementation in the health service is the current focus of ongoing implementation research.

A Case of Tumor-Induced Osteomalacia: Finding the Culprit Acetabular Tumor and Successful Resection with a Novel Hip Joint-Preserving Surgery.

INTRODUCTION: Tumor-induced osteomalacia (TIO) is a rare paraneoplastic disorder caused by tumors that produce fibroblast growth factor 23 (FGF23) resulting in phosphate wasting and inadequate bone mineralization. Complete resection of the tumor can be curative. However, these tumors are typically difficult to find anatomically due to small size and location. CASE REPORT: We present the case of a patient who presented for evaluation of recurrent fractures and hypophosphatemia in the setting of elevated FGF23 suggestive of TIO. 68Gallium-DOTATATE revealed multiple somatostatin avid lesions in several ribs, left acetabulum, sacrum, right tibia, and feet, some of which appeared with fracture on computed tomography scan, initially concerning for metastatic disease. However, the lesion in acetabulum was considered the culprit tumor given its remarkably higher maximum standard uptake values. Complete surgical removal of the FGF23-secreting tumor led to cure of this disease. CONCLUSION: This case report highlights the challenges with functional imaging differentiating fractures from the culprit lesion and reports on a novel surgical technique that allowed for surgical cure while preserving the hip joint.

From "stuck" to satisfied: Aboriginal people's experience of culturally safe care with interpreters in a Northern Territory hospital.

BACKGROUND: Globally, interpreters are underused by health providers in hospitals, despite 40 years of evidence documenting benefits to both patients and providers. At Royal Darwin Hospital, in Australia's Northern Territory, 60-90% of patients are Aboriginal, and 60% speak an Aboriginal language, but only approximately 17% access an interpreter. Recognising this system failure, the NT Aboriginal Interpreter Service and Royal Darwin Hospital piloted a new model with interpreters embedded in a renal team during medical ward rounds for 4 weeks in 2019. METHODS: This research was embedded in a larger Participatory Action Research study examining cultural safety and communication at Royal Darwin Hospital. Six Aboriginal language speaking patients (five Yolŋu and one Tiwi), three non-Indigenous doctors and five Aboriginal interpreter staff were purposefully sampled. Data sources included participant interviews conducted in either the patient's language or English, researcher field notes from shadowing doctors, doctors' reflective journals, interpreter job logs and patient language lists. Inductive narrative analysis, guided by critical theory and Aboriginal knowledges, was conducted. RESULTS: The hospital experience of Yolŋu and Tiwi participants was transformed through consistent access to interpreters who enabled patients to express their clinical and non-clinical needs. Aboriginal language-speaking patients experienced a transformation to culturally safe care. After initially reporting feeling "stuck" and disempowered when forced to communicate in English, participants reported feeling satisfied with their care and empowered by consistent access to the trusted interpreters, who shared their culture and worldviews. Interpreters also enabled providers to listen to concerns and priorities expressed by patients, which resulted in holistic care to address social determinants of health. This improved patient trajectories and reduced self-discharge rates. CONCLUSIONS: A culturally unsafe system which restricted people's ability to receive equitable healthcare in their first language was overturned by embedding interpreters in a renal medical team. This research is the first to demonstrate the importance of consistent interpreter use for providing culturally safe care for Aboriginal patients in Australia.

Sulfate transport mutants affect hydrogen sulfide and sulfite production during alcoholic fermentation.

Hydrogen sulfide is a common wine fault, with a rotten-egg odour, which is directly related to yeast metabolism in response to nitrogen and sulfur availability. In grape juice, sulfate is the most abundant inorganic sulfur compound, which is taken up by yeast through two high-affinity sulfate transporters, Sul1p and Sul2p, and a low affinity transporter, Soa1p. Sulfate contributes to H2 S production under nitrogen limitation, by being reduced via the Sulfur Assimilation Pathway (SAP). Therefore, yeast strains with limited H2 S are highly desirable. We report on the use of toxic analogues of sulfate following ethyl methane sulfate treatment, to isolate six wine yeast mutants that produce no or reduced H2 S and SO2 during fermentation in synthetic and natural juice. Four amino acid substitutions (A99V, G380R, N588K and E856K) in Sul1p were found in all strains except D25-1 which had heterozygous alleles. Two changes were also identified in Sul2p (L268S and A470T). The Sul1p (G380R) and Sul2p (A470T) mutations were chosen for further investigation as these residues are conserved amongst SLC26 membrane proteins (including sulfate permeases). The mutations were introduced into EC1118 using Crispr cas9 technology and shown to reduce accumulation of H2 S and do not result in increased SO2 production during fermentation of model medium (chemically defined grape juice) or Riesling juice. The Sul1p (G380R) and Sul2p (A470T) mutations are newly reported as causal mutations. Our findings contribute to knowledge of the genetic basis of H2 S production as well as the potential use of these strains for winemaking and in yeast breeding programmes.

Capturing yeast associated with grapes and spontaneous fermentations of the Negro Saurí minority variety from an experimental vineyard near León.

'Microbial terroir' relates to the influence of autochthonous yeasts associated with a grape cultivar on the resultant wine. Geographic region, vineyard site and topography, climate and vintage influence the biodiversity of these microbial communities. Current research focus attempts to correlate their 'microbial fingerprint' to the sensorial and chemical characteristics of varietal wines from distinct geographical wine regions. This study focuses on the minor red grape variety, Negro Saurí, which has seen a resurgence in the León Appellation of Origin in Spain as a varietal wine. An experimental vineyard at Melgarajo S.A. (42° 15' 48.68_N 5° 9' 56.66_W) was sampled over four consecutive vintages, with autochthonous yeasts being isolated from grapes, must and pilot-scale un-inoculated fermentations, and identified by ITS sequencing. Forty-nine isolates belonging to Metschnikowia pulcherrima, Lachancea thermotolerans, Hanseniaspora uvarum and Torulaspora delbrueckii were isolated from grapes and must, and early stages of fermentation dependent on seasonal variation. Saccharomyces cerevisiae predominated throughout fermentation, as a heterogeneous and dynamic population, with seven major biotypes identified amongst 110 isolates across four consecutive vintages. Twenty-four S. cerevisiae isolates representing five strains dominated in two or more vintages. Their persistence through fermentation warrants further validation of their oenological properties as starter cultures.

Flexible RSV Prefusogenic Fusion Glycoprotein Exposes Multiple Neutralizing Epitopes that May Collectively Contribute to Protective Immunity.

Human respiratory syncytial virus (RSV) is a cause of lower respiratory tract infection in infants, young children, and older adults. There is no licensed vaccine and prophylactic treatment options are limited. The RSV fusion (F) glycoprotein is a target of host immunity and thus a focus for vaccine development. F-trimers are metastable and undergo significant rearrangements from the prefusion to a stable postfusion structure with neutralizing epitopes on intermediate structures. We hypothesize that vaccine strategies that recapitulate the breathable F quaternary structure, and provide accessibility of B-cells to epitopes on intermediate conformations, may collectively contribute to protective immunity, while rigid prefusion F structures restrict access to key protective epitopes. To test this hypothesis, we used the near full-length prefusogenic F as a backbone to construct three prefusion F variants with substitutions in the hydrophobic head cavity: (1) disulfide bond mutant (DS), (2) space filling hydrophobic amino acid substitutions (Cav1), and (3) DS, Cav1 double mutant (DS-Cav1). In this study, we compared the immunogenicity of prefusogenic F to prefusion F variants in two animal models. Native prefusogenic F was significantly more immunogenic, producing high titer antibodies to prefusogenic, prefusion, and postfusion F structures, while animals immunized with DS or DS-Cav1 produced antibodies to prefusion F. Importantly, prefusogenic F elicited antibodies that target neutralizing epitopes including prefusion-specific site zero (Ø) and V and conformation-independent neutralizing sites II and IV. Immunization with DS or DS-Cav1 elicited antibodies primarily to prefusion-specific sites Ø and V with little or no antibodies to other key neutralizing sites. Animals immunized with prefusogenic F also had significantly higher levels of antibodies that cross-neutralized RSV A and B subtypes, while immunization with DS or DS-Cav1 produced antibodies primarily to the A subtype. We conclude that breathable trimeric vaccines that closely mimic the native F-structure, and incorporate strategies for B-cell accessibility to protective epitopes, are important considerations for vaccine design. F structures locked in a single conformation restrict access to neutralizing epitopes that may collectively contribute to destabilizing F-trimers important for broad protection. These results also have implications for vaccine strategies targeting other type 1 integral membrane proteins.

Evaluation of indigenous non-Saccharomyces yeasts isolated from a South Australian vineyard for their potential as wine starter cultures.

The use of non-Saccharomyces yeast in conjunction with Saccharomyces cerevisiae in wine fermentation is a growing trend in the wine industry. Non-Saccharomyces, through their distinctive production of secondary metabolites, have the potential to positively contribute to wine sensory profile. To discover new candidate strains for development as starter cultures, indigenous non-Saccharomyces were isolated from un-inoculated fermenting Shiraz musts from a South Australian vineyard (McLaren Vale wine region) and characterised. Among the 77 isolates, 7 species belonging to 5 genera (Kazachstania, Aureobasidium, Meyerozyma, Wickerhamomyces and Torulaspora) were identified by sequencing the internal transcribed spacer regions of the 5.8S rRNA gene (ITS1-5.8S-ITS2 region). The indigenous isolates were evaluated for oenological properties, namely, ethanol tolerance, enzyme activity, and H2S production. To determine their potential industrial use as starter cultures, representative isolates of each species were assessed in a sterile chemically defined grape juice and Viognier grape juice to evaluate their contribution to fermentation kinetics and production of key metabolites, including volatile compounds.

Iterative synthetic strategies and gene deletant experiments enable the first identification of polysulfides in Saccharomyces cerevisiae.

New evidence on the role of H2S as a gasotransmitter suggests that the true signalling effectors are polysulfides. Both oxidized polysulfides and hydropolysulfides were synthesized and their presence in S. cerevisiae was observed for the first time. A single gene-deletant approach allowed observation of the modulation of polysulfide species and levels.

Managing Infusion Reactions to New Monoclonal Antibodies in Multiple Myeloma: Daratumumab and Elotuzumab.

Monoclonal antibodies (elotuzumab and daratumumab) are the newest class of drugs that have proven to be efficacious antimyeloma agents. Although daratumumab, a CD38 monoclonal antibody, has established its efficacy as a single agent and in combination with immunomodulatory agents and proteasome inhibitors, elotuzumab (signaling lymphocytic activation molecule F7 monoclonal antibody) has proven activity in combination with lenalidomide and dexamethasone. Infusion-related reactions (respiratory and nonrespiratory) seem to be a common theme of adverse events with monoclonal antibodies, although the relative incidence differs across these two agents. Identifying the appropriate pre- and postinfusion medication strategies can help lower the rates of infusion-related reactions and facilitate reduction in infusion times. In this article, we review the incidence of the infusion-related reactions with elotuzumab and daratumumab and their clinical activity in myeloma, review our institutional experience of management of infusion-related reactions, and provide some practical mitigation strategies to reduce their incidence.

Roles of the genomic sequence surrounding the stem-loop structure in the junction region including the 3' terminus of open reading frame 1 in hepatitis E virus replication.

Hepatitis E virus (HEV), a member of the family Hepeviridae, causes both acute and chronic viral hepatitis. We have previously demonstrated that the stem-loop structure in the junction region (JR) of HEV genome plays a critical role in HEV replication. However, the function of the sequence bordering the JR, including the 3' terminus of open reading frame (ORF1), in HEV replication is unknown. In this study, a panel of HEV Renilla luciferase (Rluc) replicons containing various deletions at 5' or 3' termini of the JR was constructed to determine the effect of the deletions on HEV replication in Huh7 human liver cells. We showed that even a single nucleotide deletion at the 5' terminus of the JR abolished HEV replication, whereas deletions at the 3' terminus of the JR also decreased virus replication efficiency. Furthermore, we also constructed firefly luciferase and Rluc dual-reporter HEV replicons containing the 3' terminal ORF1 of various lengths and the JR inserted upstream of the Rluc reporter. A higher level of HEV replication was observed in cells transfected with replicons containing the 3' terminal ORF1 than that of the JR only replicon. We also showed that the ORF3 noncoding sequence along with the JR promoted a higher level of translation activity than that promoted by JR and the ORF2 noncoding sequence.