Inducible expression of human papillomavirus-16 L1 capsomeres in the plastomes of Nicotiana tabacum: Transplastomic plants develop normal flowers and pollen.

Human papillomavirus type-16 (HPV-16) is the major HPV type involved in causing cervical cancer among women. The disease burden is high in developing and underdeveloped countries. Previously, the constitutive expression of HPV-16 L1 protein led to male sterility in transplastomic tobacco plants. Here, the HPV-16 L1 gene was expressed in chloroplasts of Nicotiana tabacum under the control of an ethanol-inducible promoter, trans-activated by nucleus-derived signal peptide. Plants containing nuclear component were transformed with transformation vector pEXP-T7-L1 by biolistic gun. The transformation and homoplasmic status of transformed plants was verified by polymerase chain reaction and Southern blotting, respectively. Protein was induced by spraying 5% ethanol for 7 consecutive days. The correct folding of L1 protein was confirmed by antigen-capture ELISA using a conformation-specific antibody. The L1 protein accumulated up to 3 µg/g of fresh plant material. The L1 protein was further purified using affinity chromatography. All transplastomic plants developed normal flowers and produced viable seeds upon self-pollination. Pollens also showed completely normal structure under light microscope and scanning electron microscopy. These data confirm the use of the inducible expression as plant-safe approach for expressing transgenes in plants, especially those genes that cause detrimental effects on plant growth and morphology.

Physical therapy interventions for older people with vertigo, dizziness and balance disorders addressing mobility and participation: a systematic review.

BACKGROUND: Vertigo, dizziness and balance disorders (VDB) are among the most relevant contributors to the burden of disability among older adults living in the community and associated with immobility, limitations of activities of daily living and decreased participation. The aim of this study was to identify the quality of evidence of physical therapy interventions that address mobility and participation in older patients with VDB and to characterize the used primary and secondary outcomes. METHODS: A systematic search via MEDLINE (PubMed), Cochrane Library, CINAHL, PEDro, forward citation tracing and hand search was conducted initially in 11/2017 and updated in 7/2019. We included individual and cluster-randomized controlled trials and trials with quasi-experimental design, published between 2007 and 2017/2019 and including individuals ≥65 years with VDB. Physical therapy and related interventions were reviewed with no restrictions to outcome measurement. Screening of titles, abstracts and full texts, data extraction and critical appraisal was conducted by two independent researchers. The included studies were heterogeneous in terms of interventions and outcome measures. Therefore, a narrative synthesis was conducted. RESULTS: A total of 20 randomized and 2 non-randomized controlled trials with 1876 patients met the inclusion criteria. The included studies were heterogeneous in terms of complexity of interventions, outcome measures and methodological quality. Vestibular rehabilitation (VR) was examined in twelve studies, computer-assisted VR (CAVR) in five, Tai Chi as VR (TCVR) in three, canal repositioning manoeuvres (CRM) in one and manual therapy (MT) in one study. Mixed effects were found regarding body structure/function and activities/participation. Quality of life and/or falls were assessed, with no differences between groups. VR is with moderate quality of evidence superior to usual care to improve balance, mobility and symptoms. CONCLUSION: To treat older individuals with VDB, VR in any variation and in addition to CRMs seems to be effective. High-quality randomized trials need to be conducted to inform clinical decision making. TRIAL REGISTRATION: PROSPERO 2017 CRD42017080291 .

Metabolic implication of tigecycline as an efficacious second-line treatment for sorafenib-resistant hepatocellular carcinoma.

Sorafenib represents the current standard of care for patients with advanced-stage hepatocellular carcinoma (HCC). However, acquired drug resistance occurs frequently during therapy and is accompanied by rapid tumor regrowth after sorafenib therapy termination. To identify the mechanism of this therapy-limiting growth resumption, we established robust sorafenib resistance HCC cell models that exhibited mitochondrial dysfunction and chemotherapeutic crossresistance. We found a rapid relapse of tumor cell proliferation after sorafenib withdrawal, which was caused by renewal of mitochondrial structures alongside a metabolic switch toward high electron transport system (ETS) activity. The translation-inhibiting antibiotic tigecycline impaired the biogenesis of mitochondrial DNA-encoded ETS subunits and limited the electron acceptor turnover required for glutamine oxidation. Thereby, tigecycline prevented the tumor relapse in vitro and in murine xenografts in vivo. These results offer a promising second-line therapeutic approach for advanced-stage HCC patients with progressive disease undergoing sorafenib therapy or treatment interruption due to severe adverse events.

Inhibition of Cyclin-Dependent Kinase 5: A Strategy to Improve Sorafenib Response in Hepatocellular Carcinoma Therapy.

Therapeutic options for patients with advanced-stage hepatocellular carcinoma (HCC) are very limited. The only approved first-line treatment is the multi-tyrosine kinase inhibitor sorafenib, which shows low response rates and severe side effects. In particular, the compensatory activation of growth factor receptors leads to chemoresistance and limits the clinical impact of sorafenib. However, combination approaches to improve sorafenib have failed. Here we investigate the inhibition of cyclin-dependent kinase 5 (Cdk5) as a promising combination strategy to improve sorafenib response in HCC. Combination of sorafenib with Cdk5 inhibition (genetic knockdown by short hairpin RNA or CRISPR/Cas9 and pharmacologic inhibition) synergistically impaired HCC progression in vitro and in vivo by inhibiting both tumor cell proliferation and migration. Importantly, these effects were mediated by a mechanism for Cdk5: A liquid chromatography-tandem mass spectrometry-based proteomic approach revealed that Cdk5 inhibition interferes with intracellular trafficking, a process crucial for cellular homeostasis and growth factor receptor signaling. Cdk5 inhibition resulted in an accumulation of enlarged vesicles and respective cargos in the perinuclear region, considerably impairing the extent and quality of growth factor receptor signaling. Thereby, Cdk5 inhibition offers a comprehensive approach to globally disturb growth factor receptor signaling that is superior to specific inhibition of individual growth factor receptors. Conclusion: Cdk5 inhibition represents an effective approach to improve sorafenib response and to prevent sorafenib treatment escape in HCC. Notably, Cdk5 is an addressable target frequently overexpressed in HCC, and with Dinaciclib, a clinically tested Cdk5 inhibitor is readily available. Thus, our study provides evidence for clinically evaluating the combination of sorafenib and Dinaciclib to improve the therapeutic situation for patients with advanced-stage HCC.

Manual lymphatic drainage and quality of life in patients with lymphoedema and mixed oedema: a systematic review of randomised controlled trials.

PURPOSE: To assess the impact of manual lymphatic drainage (MLD) on the health-related quality of life (HRQoL) of adults with lymphoedema or mixed oedema, through a systematic review of randomised controlled trials (RCTs). METHODS: MEDLINE, EMBASE, PsycINFO, CENTRAL, the Cochrane Database of Systematic Reviews and ClinicalTrials.gov were searched to identify RCTs evaluating HRQoL after a MLD intervention compared to non-MLD interventions (PROSPERO 2016:CRD42016042255). We extracted the effect of the interventions on the HRQoL (primary outcome) as well as data on volume and functional changes, and adverse events when available (secondary outcomes). RESULTS: Eight studies were eligible. The studies were heterogeneous in the aetiology of oedema, schemes of MLD applied, additional treatments offered with MLD, length of follow-up, instruments used to assess HRQoL and interventions offered to the control group. Five studies included patients with breast cancer-related arm lymphoedema; one study reported increased HRQoL among patients randomised to the MLD group. The two RCTs that involved patients with leg mixed oedema due to chronic venous insufficiency did not find between-group differences in the overall HRQoL. One trial included patients with hand oedema from systemic sclerosis and showed higher HRQoL in the group that received MLD. No studies reported reductions in HRQoL, or severe adverse events after MLD. The small numbers of patients analysed in all studies may have resulted in lack of power to detect between-group differences in HRQoL. CONCLUSIONS: The effect of MLD on the HRQoL of patients with chronic oedema is unclear.

Clonal Evolution and Blast Crisis Correlate with Enhanced Proteolytic Activity of Separase in BCR-ABL b3a2 Fusion Type CML under Imatinib Therapy.

Unbalanced (major route) additional cytogenetic aberrations (ACA) at diagnosis of chronic myeloid leukemia (CML) indicate an increased risk of progression and shorter survival. Moreover, newly arising ACA under imatinib treatment and clonal evolution are considered features of acceleration and define failure of therapy according to the European LeukemiaNet (ELN) recommendations. On the basis of 1151 Philadelphia chromosome positive chronic phase patients of the randomized CML-study IV, we examined the incidence of newly arising ACA under imatinib treatment with regard to the p210BCR-ABL breakpoint variants b2a2 and b3a2. We found a preferential acquisition of unbalanced ACA in patients with b3a2 vs. b2a2 fusion type (ratio: 6.3 vs. 1.6, p = 0.0246) concurring with a faster progress to blast crisis for b3a2 patients (p = 0.0124). ESPL1/Separase, a cysteine endopeptidase, is a key player in chromosomal segregation during mitosis. Separase overexpression and/or hyperactivity has been reported from a wide range of cancers and cause defective mitotic spindles, chromosome missegregation and aneuploidy. We investigated the influence of p210BCR-ABL breakpoint variants and imatinib treatment on expression and proteolytic activity of Separase as measured with a specific fluorogenic assay on CML cell lines (b2a2: KCL-22, BV-173; b3a2: K562, LAMA-84). Despite a drop in Separase protein levels an up to 5.4-fold increase of Separase activity under imatinib treatment was observed exclusively in b3a2 but not in b2a2 cell lines. Mimicking the influence of imatinib on BV-173 and LAMA-84 cells by ESPL1 silencing stimulated Separase proteolytic activity in both b3a2 and b2a2 cell lines. Our data suggest the existence of a fusion type-related feedback mechanism that posttranslationally stimulates Separase proteolytic activity after therapy-induced decreases in Separase protein levels. This could render b3a2 CML cells more prone to aneuploidy and clonal evolution than b2a2 progenitors and may therefore explain the cytogenetic results of CML patients.

Atorvastatin added to interferon beta for relapsing multiple sclerosis: 12-month treatment extension of the randomized multicenter SWABIMS trial.

BACKGROUND: Statins have anti-inflammatory and immunomodulatory properties in addition to lipid-lowering effects. OBJECTIVES: To report the 12-month extension of a phase II trial evaluating the efficacy, safety and tolerability of atorvastatin 40 mg/d added to interferon beta-1b (IFNB-1b) in relapsing-remitting multiple sclerosis (RRMS). METHODS: In the randomized, multicenter, parallel-group, rater-blinded core study, 77 RRMS patients started IFNB-1b. At month three they were randomized 1∶1 to receive atorvastatin 40 mg/d or not in addition to IFNB-1b until month 15. In the subsequent extension study, patients continued with unchanged medication for another 12 months. Data at study end were compared to data at month three of the core study. RESULTS: 27 of 72 patients that finished the core study entered the extension study. 45 patients were lost mainly due to a safety analysis during the core study including a recruitment stop for the extension study. The primary end point, the proportion of patients with new lesions on T2-weighted images was equal in both groups (odds ratio 1.926; 95% CI 0.265-14.0007; p = 0.51). All secondary endpoints including number of new lesions and total lesion volume on T2-weighted images, total number of Gd-enhancing lesions on T1-weighted images, volume of grey and white matter, EDSS, MSFC, relapse rate, number of relapse-free patients and neutralizing antibodies did not show significant differences either. The combination therapy was well tolerated. CONCLUSIONS: Atorvastatin 40 mg/day in addition to IFNB-1b did not have any beneficial effects on RRMS compared to IFNB-1b monotherapy over a period of 24 months. TRIAL REGISTRATION: ClinicalTrials.gov NCT01111656.

Initial molecular response at 3 months may predict both response and event-free survival at 24 months in imatinib-resistant or -intolerant patients with Philadelphia chromosome-positive chronic myeloid leukemia in chronic phase treated with nilotinib.

PURPOSE: The association between initial molecular response and longer-term outcomes with nilotinib was examined. PATIENTS AND METHODS: Patients with imatinib-resistant or -intolerant chronic myeloid leukemia in chronic phase from the phase II nilotinib registration study with available postbaseline BCR-ABL1 transcript assessments were included (N = 237). RESULTS: BCR-ABL1 transcript levels (International Scale [IS]) at 3 months correlated with complete cytogenetic response (CCyR) by 24 months. Patients with BCR-ABL1 (IS) of > 1% to ≤ 10% at 3 months with nilotinib had higher cumulative incidence of CCyR by 24 months than patients with BCR-ABL1 (IS) of > 10% (53% v 16%). BCR-ABL1 (IS) at 3 months predicted major molecular response (MMR) by 24 months. Cumulative incidence of MMR by 24 months for patients with BCR-ABL1 (IS) of > 0.1% to ≤ 1%, > 1% to ≤ 10%, and > 10% was 65%, 27%, and 9%, respectively. These differences were observed for patients with or without baseline BCR-ABL1 mutations and for those with imatinib resistance or intolerance. Estimated event-free survival (EFS) rates at 24 months decreased with higher transcript levels at 3 months; patients with BCR-ABL1 (IS) of ≤ 1% had an estimated 24-month EFS rate of 82%, compared with 70% for patients with BCR-ABL1 (IS) of > 1% to ≤ 10% and 48% for patients with BCR-ABL1 (IS) of > 10%. CONCLUSION: Patients with BCR-ABL1 (IS) of > 10% at 3 months had a lower cumulative incidence of CCyR and MMR and lower rates of EFS versus patients with BCR-ABL1 (IS) of ≤ 10%. Prospective studies may determine whether close monitoring or alternative therapies are warranted for patients with minimal initial molecular response.

Phenotype severity in the bladder exstrophy-epispadias complex: analysis of genetic and nongenetic contributing factors in 441 families from North America and Europe.

OBJECTIVE: To identify genetic and nongenetic risk factors that contribute to the severity of the bladder exstrophy-epispadias complex (BEEC). STUDY DESIGN: Patients with BEEC from North America (n = 167) and Europe (n = 274) were included. The following data were collected: associated anomalies, parental age at conception, mode of conception, periconceptional folic acid supplementation, maternal risk factors during pregnancy, and environmental risk factors. The patients were divided into 3 subgroups according to phenotype severity: (i) mild, epispadias (n = 43); (ii) intermediate, classic bladder exstrophy (n = 366); and (iii) severe, cloacal exstrophy (n = 31). These subgroups then were compared with identify factors that contribute to phenotype severity. RESULTS: Males were overrepresented in all subgroups. A relatively high prevalence of cleft lip, with or without cleft palate, was observed. Maternal smoking and medical radiation during the first trimester were associated with the severe cloacal exstrophy phenotype. Compliance with periconceptional folic acid supplementation was associated with the mildest phenotype (epispadias). CONCLUSIONS: Periconceptional folic acid supplementation appears to prevent the development of the severe phenotype of BEEC.

Effects of genetically modified starch metabolism in potato plants on photosynthate fluxes into the rhizosphere and on microbial degraders of root exudates.

A high percentage of photosynthetically assimilated carbon is released into soil via root exudates, which are acknowledged as the most important factor for the development of microbial rhizosphere communities. As quality and quantity of root exudates are dependent on plant genotype, the genetic engineering of plants might also influence carbon partitioning within the plant and thus microbial rhizosphere community structure. In this study, the carbon allocation patterns within the plant-rhizosphere system of a genetically modified amylopectin-accumulating potato line (Solanum tuberosum L.) were linked to microbial degraders of root exudates under greenhouse conditions, using (13)C-CO(2) pulse-chase labelling in combination with phospholipid fatty acid (PLFA) analysis. In addition, GM plants were compared with the parental cultivar as well as a second potato cultivar obtained by classical breeding. Rhizosphere samples were obtained during young leaf developmental and flowering stages. (13)C allocation in aboveground plant biomass, water-extractable organic carbon, microbial biomass carbon and PLFA as well as the microbial community structure in the rhizosphere varied significantly between the natural potato cultivars. However, no differences between the GM line and its parental cultivar were observed. Besides the considerable impact of plant cultivar, the plant developmental stage affected carbon partitioning via the plant into the rhizosphere and, subsequently, microbial communities involved in the transformation of root exudates.

Current treatment concepts of CML.

The elucidation of the triggering molecular mechanism of chronic myeloid leukemia gave rise to the development of imatinib, a tyrosine kinase inhibitor and a prototype of target-oriented drugs. Imatinib led to impressing response and survival rates and now represents the standard therapy of CML. However, a significant proportion of patients do not tolerate or fail to respond to imatinib treatment. Alternative therapies can be offered to those patients. The particular challenge of CML patient management is to recognize an impending imatinib failure by adequate surveillance and to know about therapeutic options to prevent progression of the disease to accelerated phase or blast crisis since these are more difficult to control. Targeted therapy with second-generation tyrosine kinase inhibitors should be used in synopsis with mutational analysis and the patients' history. In this review we present current knowledge of diagnosis, monitoring and therapy strategies of patients with CML.

Templating α-helical poly(L-lysine)/polyanion complexes by nanostructured uniaxially oriented ultrathin polyethylene films.

We report a templating effect of uniaxially oriented melt-drawn polyethylene (MD-PE) films on α-helical poly(L-lysine)/poly(styrenesulfonate) (α-PLL/PSS) complexes deposited by the layer-by-layer (LBL) method. The melt-drawing process induced an MD-PE fiber texture consisting of nanoscale lamellar crystals embedded in amorphous regions on the MD-PE film surface whereby the common crystallographic c axis is the PE molecular chain direction parallel to the uniaxial melt-drawing direction. The MD-PE film and the α-PLL/PSS deposit were analyzed by atomic force microscopy (AFM) and in situ attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) using polarized light as a complementary method. Both methods revealed that α-PLL/PSS complexes adsorbed at the MD-PE surface were anisotropic and preferentially oriented perpendicular to the crystallographic c direction of the MD-PE film. Quantitatively, from AFM image analysis and ATR-FTIR dichroism of the amide II band of the α-PLL, mean cone opening angles of 12-18° for both rodlike α-PLL and the anisotropic α-PLL/PSS complexes with respect to the PE lamellae width direction were obtained. A model for the preferred alignment of α-PLL along the protruding PE lamellae is discussed, which is based on possible hydrophobic driving forces for the minimization of surface free energy at molecular and supermolecular topographic steps of the PE surface followed by electrostatic interactions between the interconnecting PSS and the α-PLL during layer-by-layer adsorption. This study elucidates the requirements and mechanisms involved in orienting biomolecules and may open up a path for designing templates to induce directed protein adsorption and cell growth by oriented polypeptide- or protein-modified PE surfaces.

Event-related potentials in people at risk for vascular dementia.

This study examined the relationship between the integrity of cerebrovascular microcirculation, neuropsychological testing and event-related potential indices of cognitive functioning in a nonclinical group of participants being at risk for vascular dementia. Sonographic measures, magnetic resonance (MR) scans and ERPs were recorded in 30 participants treated for arterial hypertension, with no report of neurological or psychiatric disorders. As a sonographic measure of cerebral microcirculation, the arteriorvenous cerebral transit time (cTT) was recorded. While neuropsychological measures of memory functions and general mental ability functions did not show systematic correlations with the cTT and other measures of vascular pathology, a pronounced correlation was obtained between P3a latency and cTT. Participants with long cTT showed a delayed P3a. These findings suggest that the P3a is a sensitive measure for reduced cognitive functions even at early stages of cerebrovascular pathology and by this may be a valuable tool for the early identification of cognitive deficits in individuals being at risk for vascular dementia.

Hypnotherapy in radiotherapy patients: a randomized trial.

PURPOSE: To determine whether hypnotherapy reduces anxiety and improves the quality of life in cancer patients undergoing curative radiotherapy (RT). METHODS AND MATERIALS: After providing written informed consent, 69 patients were randomized between standard curative RT alone (36 controls) and RT plus hypnotherapy (33 patients). Patients in the hypnotherapy group received hypnotherapy at the intake, before RT simulation, before the first RT session, and halfway between the RT course. Anxiety was evaluated by the State-Trait Anxiety Inventory DY-1 form at six points. Quality of life was measured by the Rand Medical Outcomes Study 36-item Health Survey (SF-36) at five points. Additionally, patients answered a questionnaire to evaluate their experience and the possible benefits of this research project. RESULTS: No statistically significant difference was found in anxiety or quality of life between the hypnotherapy and control groups. However, significantly more patients in the hypnotherapy group indicated an improvement in mental (p <0.05) and overall (p <0.05) well-being. CONCLUSION: Hypnotherapy did not reduce anxiety or improve the quality of life in cancer patients undergoing curative RT. The absence of statistically significant differences between the two groups contrasts with the hypnotherapy patients' own sense of mental and overall well-being, which was significantly greater after hypnotherapy. It cannot be excluded that the extra attention by the hypnotherapist was responsible for this beneficial effect in the hypnotherapy group. An attention-only control group would be necessary to control for this effect.

Production of human papillomavirus type 16 virus-like particles in transgenic plants.

Cervical cancer is linked to infection with human papillomaviruses (HPV) and is the third most common cancer among women worldwide. There is a strong demand for the development of an HPV preventive vaccine. Transgenic plants expressing the HPV major capsid protein L1 could be a system to produce virus-like particles for prophylactic vaccination or could even be used as edible vaccines to induce an L1-specific prophylactic immune response. Here, we describe the generation of transgenic tobacco and potato plants carrying the HPV type 16 major structural gene L1 under the control of the cauliflower mosaic virus 35S promoter. All attempts to express either the original, unmodified L1 gene or an L1 gene with a codon usage optimized for expression in plants failed. Surprisingly, small amounts of the protein were detected using an L1 gene optimized for expression in human cells. However, Northern blot analysis revealed that most of the L1 transcripts were degraded. Introduction of the translational enhancer Omega derived from the tobacco mosaic virus strongly increased transcript stability and resulted in accumulation of L1 protein to approximately 0.5 to 0.2% of total soluble protein in transgenic tobacco and potato plants, respectively. The plant-derived L1 protein displayed conformation-specific epitopes and assembled into virus-like particles. Furthermore, we did not find any indications of protein modification of the L1 protein produced in plants. Plant-derived L1 was as immunogenic as L1 expressed in baculovirus-infected insect cells. Feeding of tubers from transgenic potatoes to mice induced an anti-L1 antibody response in 3 out of 24 mice, although this response was only transient in two of the mice. Our data, however, indicate that an anti-L1 response was primed in about half of the 24 animals.

In vitro versus in vivo cellulose microfibrils from plant primary wall synthases: structural differences.

Detergent extracts of microsomal fractions from suspension cultured cells of Rubus fruticosus (blackberry) were tested for their ability to synthesize in vitro sizable quantities of cellulose from UDP-glucose. Both Brij 58 and taurocholate were effective and yielded a substantial percentage of cellulose microfibrils together with (1-->3)-beta-d-glucan (callose). The taurocholate extracts, which did not require the addition of Mg(2+), were the most efficient, yielding roughly 20% of cellulose. This cellulose was characterized after callose removal by methylation analysis, electron microscopy, and electron and x-ray synchrotron diffractions; its resistance toward the acid Updegraff reagent was also evaluated. The cellulose microfibrils synthesized in vitro had the same diameter as the endogenous microfibrils isolated from primary cell walls. Both polymers diffracted as cellulose IV(I), a disorganized form of cellulose I. Besides these similarities, the in vitro microfibrils had a higher perfection and crystallinity as well as a better resistance toward the Updegraff reagent. These differences can be attributed to the mode of synthesis of the in vitro microfibrils that are able to grow independently in a neighbor-free environment, as opposed to the cellulose in the parent cell walls where new microfibrils have to interweave with the already laid polymers, with the result of a number of structural defects.