MYC Recruits SPT5 to RNA Polymerase II to Promote Processive Transcription Elongation.

The MYC oncoprotein binds to promoter-proximal regions of virtually all transcribed genes and enhances RNA polymerase II (Pol II) function, but its precise mode of action is poorly understood. Using mass spectrometry of both MYC and Pol II complexes, we show here that MYC controls the assembly of Pol II with a small set of transcription elongation factors that includes SPT5, a subunit of the elongation factor DSIF. MYC directly binds SPT5, recruits SPT5 to promoters, and enables the CDK7-dependent transfer of SPT5 onto Pol II. Consistent with known functions of SPT5, MYC is required for fast and processive transcription elongation. Intriguingly, the high levels of MYC that are expressed in tumors sequester SPT5 into non-functional complexes, thereby decreasing the expression of growth-suppressive genes. Altogether, these results argue that MYC controls the productive assembly of processive Pol II elongation complexes and provide insight into how oncogenic levels of MYC permit uncontrolled cellular growth.

The MITOCHONDRIAL TRANSCRIPT STABILITY FACTOR 4 (MTSF4) is essential for the accumulation of dicistronic rpl5-cob mRNAs in Arabidopsis thaliana.

The Arabidopsis thaliana genome harbors more than 450 nuclear genes encoding pentatricopeptide repeat (PPR) proteins that operate in the RNA metabolism of mitochondria and/or plastids. To date, the molecular function of many PPR proteins is still unknown. Here we analyzed the nucleus-encoded gene At4g19440 coding for a P-type PPR protein. Knockout of this gene interferes with normal embryo development and seed maturation. Two experimental approaches were applied to overcome lethality and to investigate the outcome of At4g19440 knockout in adult plants. These studies revealed changes in the abundance of several mitochondria-encoded transcripts. In particular, steady-state levels of dicistronic rpl5-cob RNAs were markedly reduced, whereas levels of mature ccmC and rpl2-mttB transcripts were clearly increased. Predictions according to the one repeat to one nucleotide code for PPR proteins indicate binding of the At4g19440 protein to a previously detected small RNA at the 3' termini of the dicistronic rpl5-cob transcripts. This potential interaction indicates a function of this protein in 3' end formation and stabilization of these RNA species, whereas the increase in the levels of the ccmC mRNA along with other mitochondria-encoded RNAs seems to be a secondary effect of At4g19440 knockout. Since the inactivation of At4g19440 influences the stability of several mitochondrial RNAs we call this gene MITOCHONDRIAL TRANSCRIPT STABILITY FACTOR 4 (MTSF4). This factor will be an interesting subject to study opposing effects of a single nucleus-encoded protein on mitochondrial transcript levels.

Social relationships and their impact on health-related quality of life in a long-term breast cancer survivor cohort.

BACKGROUND: Health-related quality of life (HRQOL) has become increasingly important for breast cancer survivors, but clinically relevant declines often persist for many years after treatment. This study aimed to investigate whether social relationships can mitigate or prevent this decline in HRQOL. METHODS: Data were used from the German population-based Mamma Carcinoma Risk Factor Investigation (MARIE) cohort of 2022 breast cancer cases with follow-up information for more than 15 years after diagnosis. Correlations between social integration, social support, and global health status (GHS) as an overall measure of HRQOL were analyzed, and linear regression analysis was performed with structural equation modeling. RESULTS: The majority of participants reported high levels of social integration and social support and moderate levels of GHS. Social integration 5 years after diagnosis was associated with GHS 5 years after diagnosis (ß = 1.12; 95% CI, 0.25-1.99), but no longitudinal effects were found. Social support 5 years after diagnosis was associated with better GHS 5 years (ß = 0.42; 95% CI, 0.36-0.48) and 10 years after diagnosis (ß = 0.12; 95% CI, 0.02-0.22), whereas social support 10 years after diagnosis was associated with GHS 10 years (ß = 0.29; 95% CI, 0.20-0.39) and 15 years after diagnosis (ß = 0.10; 95% CI, 0.01-0.21). CONCLUSIONS: These results confirm that social relationships positively influence HRQOL in long-term breast cancer survivors and that their association should receive more attention clinically and beyond routine care.

Diagnostic performance of additional imaging tests for staging purposes in a bicentric German series of low-risk early breast cancer patients.

PURPOSE: Low-risk early breast cancer rarely leads to the development of metastatic disease, and in these patients, additional imaging test is controversial. The aim of our study was to evaluate the conventional staging procedures in a bicentric German series of low-risk breast carcinoma patients. METHODS: Retrospective evaluation of all patients diagnosed with early, low-risk breast cancer at Saarland University Hospital and Freiburg University Hospital in 2017 was performed. Clinical patient characteristics, the number and type of additional imaging examinations, follow-up examinations, and results were evaluated. The detection rate of metastases and the rate of false-positive findings were analyzed. RESULTS: A total of 203 patients were included, with all patients received at least one additional imaging test. Initially, a total of 562 additional imaging examinations were performed: 166 chest X-rays, 169 upper abdominal ultrasounds, 199 bone scans, 27 computer tomographies (CT) chest and abdomen, and 1 CT abdomen. 6.8% of patients had abnormal findings reported, requiring 38 additional imaging examinations. One patient (0.5%) was found to have bone metastases. The rate of false-positive findings in the performed additional imaging procedures was 6.6%. CONCLUSION: Metastatic disease was detected in one of 203 patients with low-risk early breast cancer. A total of 562 examinations and additional 38 follow-up examinations were performed without detection of metastasis (this corresponds to approximately 3 examinations/patient). The rate of false-positive findings was 6.6%. The performance of additional imaging procedures for detection of distant metastases should be critically reconsidered in patients with low-risk early breast cancer.

Transcriptionally active enhancers in human cancer cells.

The growth of human cancer cells is driven by aberrant enhancer and gene transcription activity. Here, we use transient transcriptome sequencing (TT-seq) to map thousands of transcriptionally active putative enhancers in fourteen human cancer cell lines covering seven types of cancer. These enhancers were associated with cell type-specific gene expression, enriched for genetic variants that predispose to cancer, and included functionally verified enhancers. Enhancer-promoter (E-P) pairing by correlation of transcription activity revealed ~ 40,000 putative E-P pairs, which were depleted for housekeeping genes and enriched for transcription factors, cancer-associated genes, and 3D conformational proximity. The cell type specificity and transcription activity of target genes increased with the number of paired putative enhancers. Our results represent a rich resource for future studies of gene regulation by enhancers and their role in driving cancerous cell growth.

Influences on pathologic complete response in breast cancer patients after neoadjuvant chemotherapy.

PURPOSE: Pathologic complete response is associated with longer disease-free survival and better overall survival after neoadjuvant chemotherapy in breast cancer patients. We, therefore, evaluated factors influencing pathologic complete response. METHODS: Patients receiving neoadjuvant chemotherapy from 2015 to 2018 at the Saarland University Hospital were included. Patients' age, tumor stage, tumor biology, genetic mutation, recurrent cancer, discontinuation of chemotherapy, and participation in clinical trials were extracted from electronic medical records. Binary logistic regression was performed to evaluate the influence of these factors on pathologic complete response. RESULTS: Data of 183 patients were included. The median patient's age was 54 years (22-78). The median interval between diagnosis and onset of chemotherapy was 28 days (14-91); between end of chemotherapy and surgery 28 days (9-57). Sixty-two patients (34%) participated in clinical trials for chemotherapy. A total of 86 patients (47%) achieved pathologic complete response. Patient's age, genetic mutation, recurrent cancers, or discontinuation of chemotherapy (due to side effects) and time intervals (between diagnosis and onset of chemotherapy, as well as between end of chemotherapy and surgery) did not influence pathologic complete response. Patients with high Ki67, high grading, Her2 positive tumors, as well as patients participating in clinical trials for chemotherapy had a higher chance of having pathologic complete response. Patients with Luminal B tumors had a lower chance for pathologic complete response. CONCLUSION: Particularly patients with high risk cancer and patients, participating in clinical trials benefit most from chemotherapy. Therefore, breast cancer patients can be encouraged to participate in clinical trials for chemotherapy.

A step forward: Compatible and dual-inducible expression vectors for gene co-expression in Corynebacterium glutamicum.

The Gram-positive bacterium Corynebacterium glutamicum represents a promising platform for the production of amino acids, organic acids, and other bio-products. However, the availability of only few expression vectors limits its use for production purposes, using metabolic engineering approaches when co-expression of several target genes is desired. To widen the scope for co-expression, the pCG1/p15A and pBL1/colE1 replicons were employed to construct the two differentially-inducible and compatible expression vectors pRG_Duet1 and pRG_Duet2. To functionally validate these newly constructed expression vectors, target genes for easily measurable enzymes were cloned and over-expression of these genes was investigated using respective enzyme assays. Furthermore, functionality and co-existence of the pCG1-based C. glutamicum - E. coli shuttle vector pRG_Duet1 were confirmed with pBL1-based expression vectors pRG_Duet2 and pEKEx2, using co-transformation and enzyme assays. The novel shuttle expression vectors pRG_Duet1 and pRG_Duet2 are attractive additions to the existing set of vectors for co-expression studies and metabolic engineering of C. glutamicum.

Localization of a molluscan gonadotropin-releasing hormone in Aplysia californica by in situ hybridization and immunocytochemistry.

Gonadotropin-releasing hormone (GnRH) plays important roles in vertebrate reproduction. Recently, molecules structurally similar to vertebrate GnRH were discovered in mollusks, including a gastropod, Aplysia californica. As an important step toward understanding the function of A. californica GnRH (ap-GnRH), the present study examined the localization of ap-GnRH peptide and transcript in the central and peripheral tissues. Reverse transcription polymerase chain reaction (RT-PCR) revealed wide expression of ap-GnRH in all ganglia (abdominal, buccal, cerebral, and pedal ganglia) of the central nervous system (CNS) and in multiple peripheral organs. However, in situ hybridization (ISH) revealed that cells positive for ap-GnRH are detectable only in the CNS, with the pedal ganglia containing the highest number of ap-GnRH-positive neurons, followed by the cerebral and abdominal ganglia. Most neurons positive for the transcript were simultaneously positive for the peptide, although some discrepancies were observed in cerebral and abdominal ganglia. Overall, our data suggest the de novo synthesis of ap-GnRH is restricted to the CNS, with the pedal ganglia being the primary source of ap-GnRH. Our results support the notion that ap-GnRH is a bona-fide neuropeptide that may assume diverse central functions, including those unrelated to reproduction.

Presence of keel bone damage in laying hens, pullets and roosters of local chicken breeds.

In commercial laying hens, keel bone damage (KBD) is a severe health and welfare problem leading to pain, reduced mobility and decreased laying performance. Flocks of all production systems and hybrid lines can be affected. KBD is a multifactorial welfare issue and, among other factors, associated with a high laying performance which negatively affects the calcium deposit in the medullary bones. Therefore, mature hens of local breeds with much lower egg production than commercial hybrids may be expected to show less or even no keel bone damage. This study evaluates (i) the prevalence of KBD in local breeds, (ii) the difference in type and level of damages, and (iii) if roosters and pullets are also affected. In total, we palpated 343 mature hens, 40 pullets, and 18 roosters of 13 different local breeds and one commercial hybrid. The animals were kept on eight different farms in free-range or floor-housing systems. Our results showed that on average 44.2% of mature hens per local breed were affected by KBD (range: 11.1%-84.7%). We found deviation of less than 1 cm in 26.9%, deviations of more than 1 cm in 6.4% and palpable fractures in 23.8% of the mature hens of local breeds. The tip was damaged in 23.6% of the mature hens. Also, pullets and roosters were affected by KBD. Finally, we found that KBD also occurs in local breeds. Therefore, we conclude that even the low laying performance of local breeds does not prevent them from the occurrence of KBD.KBD in local breeds may rather be associated with genetics (breed) as well as management and housing. Thus, breeders of local breeds should include bone health as a selection trait. Owners of local breeds should also pay attention to the condition of the keel and ought to be trained about preventive measures.

Reinforcement of the Tumor Suppressing Properties of microRNA-1 by Substitution at the C2' Position of Varying Ribose Residues in Chemically Synthesized microRNA-1 Molecules.

BACKGROUND/AIM: Tumor suppressive microRNAs (miR) are frequently down-regulated during cancer development. The application of synthetic miR molecules restoring suppressed miR, therefore, opens up innovative possibilities in future anticancer therapy. The potential application, however, is limited by the instability of RNA molecules. The presented proof-of-principle study evaluates the potential of using synthetic chemically modified miR molecules as anticancer drugs. MATERIALS AND METHODS: Chemically synthesized miR-1 molecules containing two 2'-O-RNA modifications, 2'-O-methyl- and 2'-fluoro-derivatives, introduced at different positions of the 3'-terminus, were transfected into prostate cancer (PC) cells (LNCaP, PC-3). Detectability was measured by quantitative RT-PCR. The effect of modifications regarding the growth inhibitory activity of miR-1 was investigated by cell growth kinetics with transfected PC cells. RESULTS: All variants of synthetic modified miR-1 could be transfected into PC cells and were detectable by RT-PCR. Depending on the chemical modification, but especially on the position of the modification, the growth inhibitory activity of synthetic modified miR-1 was increased compared to synthetic unmodified miR-1. CONCLUSION: Synthetic miR-1 can be enhanced in its biological activity by modification of the C2'-OH group. This depends on the chemical substituent, the position and number of substituted nucleotides. The molecular fine-tuning of tumor suppressive miR like miR-1 may represent a promising approach for the development of multi-targeting nucleic acid-based drugs for cancer therapy.

Perturbed epigenetic transcriptional regulation in AML with IDH mutations causes increased susceptibility to NK cells.

Isocitrate dehydrogenase (IDH) mutations are found in 20% of acute myeloid leukemia (AML) patients. However, only 30-40% of the patients respond to IDH inhibitors (IDHi). We aimed to identify a molecular vulnerability to tailor novel therapies for AML patients with IDH mutations. We characterized the transcriptional and epigenetic landscape with the IDH2i AG-221, using an IDH2 mutated AML cell line model and AML patient cohorts, and discovered a perturbed transcriptional regulatory network involving myeloid transcription factors that were partly restored after AG-221 treatment. In addition, hypermethylation of the HLA cluster caused a down-regulation of HLA class I genes, triggering an enhanced natural killer (NK) cell activation and an increased susceptibility to NK cell-mediated responses. Finally, analyses of DNA methylation data from IDHi-treated patients showed that non-responders still harbored hypermethylation in HLA class I genes. In conclusion, this study provides new insights suggesting that IDH mutated AML is particularly sensitive to NK cell-based personalized immunotherapy.

An insight into the runs of homozygosity distribution and breed differentiation in Mangalitsa pigs.

Mangalitsa pigs exhibit three distinct coat color patterns based on which they are described as Red, Blond, and Swallow-bellied. The current study investigated genome-wide diversity and selection signatures in the three breeds using fixation index, runs of homozygosity and population structure analyses. The analyses were originally based on quality-controlled data on 77 Mangalitsa animals from Germany, including 23 Blond, 30 Swallow-bellied and 24 Red Mangalitsa genotyped with a customized version of the ProcineSNP60 v2 Genotyping Bead Chip. Also, 20 Hungarian Mangalitsa genotypes were included as outgroup data for comparison. Estimates of observed heterozygosity were 0.27, 0.28, and 0.29, and inbreeding coefficients estimated based on runs of homozygosity were 24.11%, 20.82%, and 16.34% for Blond, Swallow-bellied and Red Mangalitsa, respectively. ROH islands were detected in all breeds, however, none of these were shared amongst them. The KIF16B gene previously reported to play a role in synaptic signaling was found in a ROH island (SSC17: 16-26) in Swallow-bellied Mangalitsa. The same gene was found to harbor a significantly differentiated SNP (MARC0032380) while contrasting either Blond or Red to Swallow-belied Mangalitsa. In the Red Mangalitsa, some ROH islands were associated with genes that play a role in meat quality traits, i.e., ABCA12, VIL1, PLSCR5, and USP37. Our population structure analysis highlighted a separation of the three breeds, but also showed the closest relatedness between Red and Blond Mangalitsa pigs. Findings of this study improve our understanding of the diversity in the three breeds of Mangalitsa pigs.

A tagged visual analog scale is a reliable method to assess keel bone deviations in laying hens from radiographs.

Laying hens often suffer from keel bone damage (KBD) that includes pathologies with different etiologies, like diverse forms of fractures and deviations. Since KBD is a problem in all countries and housing systems, methods for the assessment of deviations are urgently needed. Comparisons between genetic lines and between studies are important to detect underlying mechanisms. Field researchers often use palpation as a low-cost and feasible technique for the assessment of KBD. In contrast to palpation, radiography is effective and highly precise at least in detecting keel bone fractures. The aim of this study was to: i) develop a scoring system to assess keel bone deviations from radiographs, ii) to assess inter- and intra-observer reliability of this scoring system, and iii) to investigate whether fractures and deviations of the keel are correlated. In total, 192 hens were used for the investigation. Digital radiographs were taken and evaluated for all hens after slaughter. We developed a tagged visual analog scale with two extreme images as anchors and four intermediate tags, resulting in six images representing the range from "no deviation" to "highly deviated" on a 10 cm line. Eleven participants scored 50 radiographs of keels with varying degree of severity, whereas five images were scored twice to assess intra-observer reliability. Intraclass correlation coefficient for inter-observer reliability was 0.979 with a confidence interval of 0.968 < ICC < 0.987 (F49,268 = 54.2, p < 0.0001). Intraclass correlation coefficient for intra-observer reliability was 0.831 with a confidence interval of 0.727 < ICC < 0.898 (F54,55 = 10.8, p < 0.0001). Individual intra-observer reliability ranged from 0.6 to 0.949. The Spearman correlation showed a strong positive correlation of fractures and deviations (s roh= 0.803, p < 0.001). The tagged visual analog scale could be a reliable instrument for the scoring of keel bone deviations. Our results support the assumption that the majority of highly deviated keels suffer from fractures as well. Further research is needed to investigate the correlation of palpation scores with the evaluation on radiographs.

Automatic Assessment of Keel Bone Damage in Laying Hens at the Slaughter Line.

Keel bone damage (KBD) can be found in all commercial laying hen flocks with a wide range of 23% to 69% of hens/flock found to be affected in this study. As KBD may be linked with chronic pain and a decrease in mobility, it is a serious welfare problem. An automatic assessment system at the slaughter line could support the detection of KBD and would have the advantage of being standardized and fast scoring including high sample sizes. A 2MP stereo camera combined with an IDS imaging color camera was used for the automatic assessment. A trained human assessor visually scored KBD in defeathered hens during the slaughter process and compared results with further human assessors and automatic recording. In a first step, an algorithm was developed on the basis of assessments of keel status of 2287 hens of different genetics with varying degrees of KBD. In two optimization steps, performance data were calculated, and flock prevalences were determined, which were compared between the assessor and the automatic system. The proposed technique finally reached a sensitivity of 0.95, specificity of 0.77, accuracy of 0.86 and precision of 0.81. In the last optimization step, the automatic system scored on average about 10.5% points lower KBD prevalences than the human assessor. However, a proposed change of scoring system (setting the limit for KBD at 0.5 cm deviation from the straight line) would lower this deviation. We conclude that the developed automatic scoring technique is a reliable and potentially valuable tool for the assessment of KBD.

Status of Sentinel Lymph Node Biopsy in Endometrial Cancer.

The role of lymphadenectomy in surgical staging remains one of the biggest controversies in the management of endometrial cancer. The concept of sentinel lymph node biopsy in endometrial cancer has been evaluated for a number of years, with promising sensitivity rates and negative predictive values. The possibility of adequate staging while avoiding systematic lymphadenectomy leads to a significant reduction in the rate of peri- and postoperative morbidity. Nevertheless, the status of sentinel lymph node biopsy in endometrial cancer has not yet been fully elucidated and is variously assessed internationally. According to current European guidelines and recommendations, sentinel lymph node biopsy in endometrial cancer should be performed only in the context of clinical studies. In this review article, the developments of the past decade are explored concisely. In addition, current data regarding the technical aspects, accuracy and prognostic relevance of sentinel lymph node biopsy are explained and evaluated critically.

Status of Sentinel Lymph Node Biopsy in Vulvar and Cervical Cancer.

Assessment of lymphatic metastasis is an essential component of solid tumour staging. Sentinel lymph node (SLN) biopsy is a minimally invasive procedure that allows regional lymph node involvement by tumour to be estimated by selectively examining the sentinel lymph node while minimising the morbidity of systematic lymph node dissection. Within the group of genital cancers, the diagnostic value of SLN biopsy is rated differently. For selected patients with early-stage vulvar cancer (unifocal primary tumour < 4 cm, clinically negative inguinal lymph nodes) the SLN technique is already an established procedure in the guidelines of the German Society for Gynaecology and Obstetrics (DGGG)/German Cancer Society (DKG) and the recommendations of the European Society of Gynaecological Oncology (ESGO). For cervical cancer, SLN biopsy has not yet been sufficiently standardised but can be considered for patients without risk factors with a primary tumour size < 2 cm. The SLN is identified by combined use of radioactive 99m technetium nanocolloid and patent blue. The use of indocyanine green offers an alternative for SLN identification with few side effects. Recent studies aim to increase the diagnostic reliability of intraoperative frozen section analysis as this continues to show limited sensitivity in both vulvar and cervical cancer. The rate of detection of micrometastases can be increased by additional ultrastaging, the prognostic significance of which for both diseases is still unclear. The prognostic value of SLN biopsy compared with systematic lymph node dissection is being investigated in current studies (GROINSS-V-II for vulvar cancer and SENTIX-, SENTICOL-3 for cervical cancer). For this review article, a guideline-based literature search was performed in the National Library of Medicine (PubMed/MEDLINE) database with a particular focus on recent cohort studies and conference contributions.

Husbandry Conditions and Welfare Outcomes in Organic Egg Production in Eight European Countries.

In the European research project HealthyHens, welfare indicators as well as husbandry and management conditions were recorded in 107 organic laying hen farms in eight countries. Farms were visited at peak and end of lay. Egg production was on average comparable to breeder specifications. A mean mortality of 5.7% and mean prevalences of footpad lesions of 30.5%, keel bone damage of 44.5%, 57.3% of flocks with on average >200 Ascarid eggs per gram faeces and 28.2% of flocks with >100 mites/trap were recorded. A large variation between flocks indicated options for improvement. Based on the results, the following measures can be recommended: (i) decreasing mite and worm infestation and (ii) providing an attractive covered veranda, because of their association with decreased mortality; (iii) maximising access to the free range, because of its relation to decreased A. galli infection and less injurious pecking; (iv) feeding sufficient protein levels and (v) providing adequate litter as preventive measure against feather pecking and cannibalism; (vi) ensuring that the birds have sufficient weight and (vii) preventing accidents by adequate hen house facilities and light conditions to reduce keel bone damage. These primarily management-based measures have the potential to improve bird welfare both in terms of behavioural and health aspects.

NASC-seq monitors RNA synthesis in single cells.

Sequencing of newly synthesised RNA can monitor transcriptional dynamics with great sensitivity and high temporal resolution, but is currently restricted to populations of cells. Here, we develop new transcriptome alkylation-dependent single-cell RNA sequencing (NASC-seq), to monitor newly synthesised and pre-existing RNA simultaneously in single cells. We validate the method on pre-labelled RNA, and by demonstrating that more newly synthesised RNA was detected for genes with known high mRNA turnover. Monitoring RNA synthesis during Jurkat T-cell activation with NASC-seq reveals both rapidly up- and down-regulated genes, and that induced genes are almost exclusively detected as newly transcribed. Moreover, the newly synthesised and pre-existing transcriptomes after T-cell activation are distinct, confirming that NASC-seq simultaneously measures gene expression corresponding to two time points in single cells. Altogether, NASC-seq enables precise temporal monitoring of RNA synthesis at single-cell resolution during homoeostasis, perturbation responses and cellular differentiation.

Different promoter affinities account for specificity in MYC-dependent gene regulation.

Enhanced expression of the MYC transcription factor is observed in the majority of tumors. Two seemingly conflicting models have been proposed for its function: one proposes that MYC enhances expression of all genes, while the other model suggests gene-specific regulation. Here, we have explored the hypothesis that specific gene expression profiles arise since promoters differ in affinity for MYC and high-affinity promoters are fully occupied by physiological levels of MYC. We determined cellular MYC levels and used RNA- and ChIP-sequencing to correlate promoter occupancy with gene expression at different concentrations of MYC. Mathematical modeling showed that binding affinities for interactions of MYC with DNA and with core promoter-bound factors, such as WDR5, are sufficient to explain promoter occupancies observed in vivo. Importantly, promoter affinity stratifies different biological processes that are regulated by MYC, explaining why tumor-specific MYC levels induce specific gene expression programs and alter defined biological properties of cells.