Axillary Staging after Neoadjuvant Chemotherapy for Initially Node-Positive Breast Carcinoma in Germany: Initial Data from the AXSANA study.

Introduction To date, the optimal axillary staging procedure for initially node-positive breast carcinoma patients after neoadjuvant chemotherapy (NACT) has been unclear. The aim of the AXSANA study is to prospectively compare different surgical staging techniques with respect to the oncological outcome and quality of life for the patients. Little is known about current clinical practice in Germany. Material and Methods In this paper we analyzed data from patients enrolled in the AXSANA study at German study sites from June 2020 to March 2022. Results During the period under investigation, 1135 patients were recruited at 143 study sites. More than three suspicious lymph nodes were initially found in 22% of patients. The target lymph node (TLN) was marked in 64% of cases. This was done with clips/coils in 83% of patients, with magnetic seeds or carbon suspension in 8% each, and with a radar marker in 1% of patients. After NACT, targeted axillary dissection (TAD) or axillary lymphadenectomy (ALND) were each planned in 48% of patients, and sentinel lymph node biopsy alone (SLNB) in 2%. Clinically, the nodal status after NACT was found to be unremarkable in 65% of cases. Histological lymph node status was correctly assessed by palpation in 65% of patients and by sonography in 69% of patients. Conclusion At the German AXSANA study sites, TAD and ALND are currently used as the most common surgical staging procedures after NACT in initially node-positive breast cancer patients. The TLN is marked with various markers prior to NACT. Given the inadequate accuracy of clinical assessment of axillary lymph node status after NACT, it should be questioned whether axillary dissection after NACT should be performed based on clinical assessment of nodal status alone.

Quantification of Isomaltulose in Food Products by Using Heteronuclear Single Quantum Coherence NMR-Experiments.

Isomaltulose is a commonly used sweetener in sports nutrition and in products intended for consumption by diabetics. Because previously established chromatographic methods for quantification of isomaltulose suffer from long analysis times (60-210 min), faster quantitative approaches are required. Here, an HSQC (heteronuclear single quantum coherence) experiment with reduced interscan delay was established in order to quantify isomaltulose next to potential additional sugars such as d-glucose, d-fructose, d-galactose, sucrose, lactose, and maltose in 53 min. By using HSQC coupled to non-uniform sampling (NUS) as well as ASAP-HSQC (acceleration by sharing adjacent polarization), analysis times were reduced to a few minutes. Application of NUS-HSQC with reduced interscan delay takes 27 min, resulting in accurate and precise data. In principle, application of ASAP-HSQC approaches (with analysis times as low as 6 min) can be used; however, precision data may not suffice all applications.

Positive Microbiological Cultures in the Respiratory Tract of High Model for End-Stage Liver Disease (MELD) Liver Transplant Recipients With and Without Pneumonia.

BACKGROUND: Pneumonia in liver transplant recipients is one of the most common infections in the early phase after transplantation. The diagnosis is based on clinical signs combined with positive microbiological samples taken from the lower respiratory tract. However, the role of bacterial colonization is not clear, nor is its association with pneumonia or its long-term consequences. The aim of this study was to investigate the association between positive microbiological findings and clinically relevant pneumonia and analyze different clinical and laboratory parameters for their association with pneumonia in liver transplant recipients. METHODS: This was a retrospective analysis of 266 adult orthotopic liver transplantations between January 2008 and December 2013. A multidisciplinary in-house specialist panel established and confirmed the diagnosis of clinically relevant pneumonia in microbiologically positive patients. RESULTS: Of the 266 transplantations analyzed, 54 patients (20%) showed microbiologically positive trachea-bronchial cultures during the first 21 days after liver transplantation. Of those 54 patients, 24 (44.4%) had pneumonia as rated by the multidisciplinary specialist panel. Presence of gram-negative Enterobacteriaceae (P = .013) and positive chest radiologic findings (P = .035) were associated with pneumonia in microbiological-positive patients. Although patients with pneumonia had the lowest long-term survival, those without pneumonia but with positive microbiological cultures had still worse survival compared with the Model for End-Stage Liver Disease-matched control group without positive cultures (P = .012). CONCLUSIONS: Gram-negative Enterobacteriaceae and positive radiologic findings were associated with pneumonia in liver transplant recipients with positive microbiological trachea-bronchial cultures. Recipients with bacterial colonization without pneumonia also showed decreased long-term survival.

Natural Zeitgebers Under Temperate Conditions Cannot Compensate for the Loss of a Functional Circadian Clock in Timing of a Vital Behavior in Drosophila.

The adaptive significance of adjusting behavioral activities to the right time of the day seems obvious. Laboratory studies implicated an important role of circadian clocks in behavioral timing and rhythmicity. Yet, recent studies on clock-mutant animals questioned this importance under more naturalistic settings, as various clock mutants showed nearly normal diel activity rhythms under seminatural zeitgeber conditions. We here report evidence that proper timing of eclosion, a vital behavior of the fruit fly Drosophila melanogaster, requires a functional molecular clock under quasi-natural conditions. In contrast to wild-type flies, period01 mutants with a defective molecular clock showed impaired rhythmicity and gating in a temperate environment even in the presence of a full complement of abiotic zeitgebers. Although period01 mutants still eclosed during a certain time window during the day, this time window was much broader and loosely defined, and rhythmicity was lower or lost as classified by various statistical measures. Moreover, peak eclosion time became more susceptible to variable day-to-day changes of light. In contrast, flies with impaired peptidergic interclock signaling (Pdf01 and han5304 PDF receptor mutants) eclosed mostly rhythmically with normal gate sizes, similar to wild-type controls. Our results suggest that the presence of natural zeitgebers is not sufficient, and a functional molecular clock is required to induce stable temporal eclosion patterns in flies under temperate conditions with considerable day-to-day variation in light intensity and temperature. Temperate zeitgebers are, however, sufficient to functionally rescue a loss of PDF-mediated clock-internal and -output signaling.

WEclMon - A simple and robust camera-based system to monitor Drosophila eclosion under optogenetic manipulation and natural conditions.

Eclosion in flies and other insects is a circadian-gated behaviour under control of a central and a peripheral clock. It is not influenced by the motivational state of an animal, and thus presents an ideal paradigm to study the relation and signalling pathways between central and peripheral clocks, and downstream peptidergic regulatory systems. Little is known, however, about eclosion rhythmicity under natural conditions, and research into this direction is hampered by the physically closed design of current eclosion monitoring systems. We describe a novel open eclosion monitoring system (WEclMon) that allows the puparia to come into direct contact with light, temperature and humidity. We demonstrate that the system can be used both in the laboratory and outdoors, and shows a performance similar to commercial closed funnel-type monitors. Data analysis is semi-automated based on a macro toolset for the open imaging software Fiji. Due to its open design, the WEclMon is also well suited for optogenetic experiments. A small screen to identify putative neuroendocrine signals mediating time from the central clock to initiate eclosion showed that optogenetic activation of ETH-, EH and myosuppressin neurons can induce precocious eclosion. Genetic ablation of myosuppressin-expressing neurons did, however, not affect eclosion rhythmicity.

Central and peripheral clocks are coupled by a neuropeptide pathway in Drosophila.

Animal circadian clocks consist of central and peripheral pacemakers, which are coordinated to produce daily rhythms in physiology and behaviour. Despite its importance for optimal performance and health, the mechanism of clock coordination is poorly understood. Here we dissect the pathway through which the circadian clock of Drosophila imposes daily rhythmicity to the pattern of adult emergence. Rhythmicity depends on the coupling between the brain clock and a peripheral clock in the prothoracic gland (PG), which produces the steroid hormone, ecdysone. Time information from the central clock is transmitted via the neuropeptide, sNPF, to non-clock neurons that produce the neuropeptide, PTTH. These secretory neurons then forward time information to the PG clock. We also show that the central clock exerts a dominant role on the peripheral clock. This use of two coupled clocks could serve as a paradigm to understand how daily steroid hormone rhythms are generated in animals.

Niche WNT5A regulates the actin cytoskeleton during regeneration of hematopoietic stem cells.

Here, we show that the Wnt5a-haploinsufficient niche regenerates dysfunctional HSCs, which do not successfully engraft in secondary recipients. RNA sequencing of the regenerated donor Lin- SCA-1+ KIT+ (LSK) cells shows dysregulated expression of ZEB1-associated genes involved in the small GTPase-dependent actin polymerization pathway. Misexpression of DOCK2, WAVE2, and activation of CDC42 results in apolar F-actin localization, leading to defects in adhesion, migration and homing of HSCs regenerated in a Wnt5a-haploinsufficient microenvironment. Moreover, these cells show increased differentiation in vitro, with rapid loss of HSC-enriched LSK cells. Our study further shows that the Wnt5a-haploinsufficient environment similarly affects BCR-ABLp185 leukemia-initiating cells, which fail to generate leukemia in 42% of the studied recipients, or to transfer leukemia to secondary hosts. Thus, we show that WNT5A in the bone marrow niche is required to regenerate HSCs and leukemic cells with functional ability to rearrange the actin cytoskeleton and engraft successfully.

Loss of Sfrp2 in the Niche Amplifies Stress-Induced Cellular Responses, and Impairs the In Vivo Regeneration of the Hematopoietic Stem Cell Pool.

Sfrp2 is overexpressed in stromal cells which maintain hematopoietic stem cells (HSCs) during in vitro culture. We here showed, that coculture of hematopoetic cells with stromal cells with reduced expression of Sfrp2 increases the number lineage-negative Kit(+) Sca-1(+) (LSK) and progenitor cells in vitro. The LSK cells from these cocultures showed activation of canonical Wnt signaling, higher levels of Ki-67, BrdU incorporation, and the number of γH2A.X positive foci. Total repopulating activity of these cultures was, however, diminished, indicating loss of HSC. To extend these in vitro data, we modelled stress in vivo, i.e., by aging, or 5-FU treatment in Sfrp2(-) (/) (-) mice, or replicative stress in regeneration of HSCs in Sfrp2(-) (/) (-) recipients. In all three in vivo stress situations, we noted an increase of LSK cells, characterized by increased levels of ß-catenin and cyclin D1. In the transplantation experiments, the increase in LSK cells in primary recipients was subsequently associated with a progressive loss of HSCs in serial transplantations. Similar to the in vitro coculture stress, in vivo genotoxic stress in 5-FU-treated Sfrp2(-) (/) (-) mice increased cell cycle activity of LSK cells with higher levels of BrdU incorporation, increased expression of Ki-67, and canonical Wnt signaling. Importantly, as noted in vitro, increased cycling of LSKs in vivo was accompanied by a defective γH2A.X-dependent DNA damage response and depolarized localization of acetylated H4K16. Our experiments support the view that Sfrp2 expression in the niche is required to maintain the HSC pool by limiting stress-induced DNA damage and attenuating canonical Wnt-mediated HSC activation. Stem Cells 2016;34:2381-2392.

Stroma-Derived Connective Tissue Growth Factor Maintains Cell Cycle Progression and Repopulation Activity of Hematopoietic Stem Cells In Vitro.

Hematopoietic stem cells (HSCs) are preserved in co-cultures with UG26-1B6 stromal cells or their conditioned medium. We performed a genome-wide study of gene expression changes of UG26-1B6 stromal cells in contact with Lineage⁻ SCA-1⁺ KIT⁺ (LSK) cells. This analysis identified connective tissue growth factor (CTGF) to be upregulated in response to LSK cells. We found that co-culture of HSCs on CTGF knockdown stroma (shCtgf) shows impaired engraftment and long-term quality. Further experiments demonstrated that CD34⁻ CD48⁻ CD150⁺ LSK (CD34⁻ SLAM) cell numbers from shCtgf co-cultures increase in G0 and senescence and show delayed time to first cell division. To understand this observation, a CTGF signaling network model was assembled, which was experimentally validated. In co-culture experiments of CD34⁻ SLAM cells with shCtgf stromal cells, we found that SMAD2/3-dependent signaling was activated, with increasing p27(Kip1) expression and downregulating cyclin D1. Our data support the view that LSK cells modulate gene expression in the niche to maintain repopulating HSC activity.