Case Report: A Sudden Thyroid-Related Death of a 15-Year-Old Girl.

A 15-year-old young girl was found dead at home. There were no indications of any intervention or the application of force. On the previous day, she was admitted to hospital because of palpitations, fatigue, a headache, and a swollen neck. During a physical examination, a swollen thyroid gland and tachycardia were found. In the family history, her mother had thyroid disease. According to the laboratory values, she had elevated thyroid hormone levels. After administration of beta-blockers, the patient was discharged and died at home during the night. The parents denounced the hospital for medical malpractice; therefore, a Forensic Autopsy was performed. Based on the available clinical data, the autopsy, histological and toxicological results, the cause of death was stated as multiorgan failure due to disseminated intravascular coagulation (DIC) caused by the autoimmune Graves disease. The forensic assessment of the case does not reveal medical malpractice. Post-mortem diagnoses of thyroid disorders in cases of sudden death can be challenging. However, as the reported case illustrates, the diagnosis could be established after a detailed evaluation of antemortem clinical data, autopsy results, histology, and a toxicological examination.

Profiling of Copy Number Alterations Using Low-Coverage Whole-Genome Sequencing Informs Differential Diagnosis and Prognosis in Primary Cutaneous Follicle Center Lymphoma.

Primary cutaneous follicle center lymphoma (PCFCL) has an excellent prognosis using local treatment, whereas nodal follicular lymphoma (nFL), occasionally presenting with cutaneous spread, often requires systemic therapy. Distinction of the 2 diseases based on histopathology alone might be challenging. Copy number alterations (CNAs) have scarcely been explored on a genome-wide scale in PCFCL; however, they might serve as potential biomarkers during differential diagnosis and risk stratification. Low-coverage whole-genome sequencing is a robust, high-throughput method for genome-wide copy number profiling. In this study, we analyzed 28 PCFCL samples from 20 patients and compared the copy number profiles with a cohort of diagnostic samples of 64 nFL patients. Although the copy number profile of PCFCL was similar to that of nFL, PCFCL lacked amplifications of 18q, with the frequency peaking at 18q21.33 in nFL cases involving the BCL2 locus (PCFCL: 5.0% vs nFL: 31.3%, P = .018, Fisher exact test). Development of distant cutaneous spread was significantly associated with higher genomic instability including the proportion of genome altered (0.02 vs 0.13, P = .033) and number of CNAs (2 vs 9 P = .017), as well as the enrichment of 2p22.2-p15 amplification involving REL and XPO1 (6.3% vs 60.0%, P = .005), 3q23-q24 amplification (0.0% vs 50.0%, P = .004), 6q16.1-q23.3 deletion (6.3% vs 50.0%, P = .018), and 9p21.3 deletion covering CDKN2A and CDKN2B loci (0.0% vs 40.0%, P = .014, all Fisher exact test) in PCFCL. Analysis of sequential tumor samples in 2 cases harboring an unfavorable clinical course pointed to the acquisition of 2p amplification in the earliest common progenitor underlining its pivotal role in malignant transformation. By performing genome-wide copy number profiling on the largest patient cohort to date, we identified distinctive CNA alterations conceivably facilitating the differential diagnosis of PCFCL and secondary cutaneous involvement of nFL and potentially aiding the risk stratification of patients with PCFCL in the future.

Low-burden TP53 mutations represent frequent genetic events in CLL with an increased risk for treatment initiation.

TP53 aberrations predict chemoresistance and represent a contraindication for the use of standard chemoimmunotherapy in chronic lymphocytic leukaemia (CLL). Recent next-generation sequencing (NGS)-based studies have identified frequent low-burden TP53 mutations with variant allele frequencies below 10%, but the clinical impact of these low-burden TP53 mutations is still a matter of debate. In this study, we aimed to scrutinise the subclonal architecture and clinical impact of TP53 mutations using a sensitive, NGS-based mutation analysis in a 'real-world' cohort of 901 patients with CLL. In total, 225 TP53 mutations were identified in 17.5% (158/901) of the patients; 48% of these alterations represented high-burden mutations, while 52% were low-burden TP53 mutations. Low-burden mutations as sole alterations were identified in 39% (62/158) of all mutated cases with 82% (51/62) of these being represented by a single low-burden TP53 mutation. Patients harbouring low-burden TP53 mutations had significantly lower time to first treatment compared to patients with wild-type TP53. Our study has expanded the knowledge on the frequency, clonal architecture, and clinical impact of low-burden TP53 mutations. By demonstrating that patients with sole low-burden TP53 variants represent more than one-third of patients with TP53 mutations and have an increased risk for treatment initiation, our findings strengthen the need to redefine the threshold of TP53 variant reporting to below 10% in the routine diagnostic setting.

Landscape of BCL2 Resistance Mutations in a Real-World Cohort of Patients with Relapsed/Refractory Chronic Lymphocytic Leukemia Treated with Venetoclax.

The oral, highly selective Bcl2 inhibitor venetoclax has substantially improved the therapeutic landscape of chronic lymphocytic leukemia (CLL). Despite the remarkable response rates in patients with relapsed/refractory (R/R) disease, acquired resistance is the leading cause of treatment failure, with somatic BCL2 mutations being the predominant genetic drivers underpinning venetoclax resistance. To assess the correlation between disease progression and the most common BCL2 mutations G101V and D103Y, sensitive (10-4) screening for the most common BCL2 mutations G101V and D103Y was performed in 67 R/R CLL patients during venetoclax single-agent or venetoclax-rituximab combination therapy. With a median follow-up time of 23 months, BCL2 G101V and D103Y were detected in 10.4% (7/67) and 11.9% (8/67) of the cases, respectively, with four patients harboring both resistance mutations. Ten out of eleven patients carrying BCL2 G101V and/or D103Y experienced relapse during the follow-up period, representing 43.5% of the cases (10/23) showing clinical signs of disease progression. All BCL2 G101V or D103Y variants were detected in patients receiving venetoclax as a continuous single-agent treatment while these mutations were not observed during or after fixed-duration venetoclax therapy. Targeted ultra-deep sequencing of BCL2 uncovered three additional variants in four patient samples obtained at relapse, suggesting convergent evolution and implying a cooperating role of BCL2 mutations in driving venetoclax resistance. This cohort is the largest R/R CLL patient population reported to date in which BCL2 resistance mutations were investigated. Our study demonstrates the feasibility and clinical value of sensitive screening for BCL2 resistance mutations in R/R CLL.

Single thin-plate compression of multi-TW laser pulses to 3.9 fs.

Post-compression of 12-fs laser pulses with multi-TW peak power from an optical parametric chirped pulse amplification (OPCPA) system was performed by using a single thin fused silica plate in a vacuum. By optimizing the input pulses in both spatial and temporal domains, after compression with customized chirped mirrors, we achieved pulses as short as 3.87 fs, in combination with 12-mJ energy. The spatio-spectral quality of the post-compressed pulses was thoroughly analyzed. The generated 1.4-cycle pulses pave the way for next generation attosecond and particle acceleration experiments.

The genetic legacy of the Hunyadi descendants.

The Hunyadi family is one of the most influential families in the history of Central Europe in the 14th-16th centuries. The family's prestige was established by Johannes Hunyadi, a Turk-beater who rose to the position of governor of the Kingdom of Hungary. His second son, Matthias Hunyadi, became the elected ruler of the Kingdom of Hungary in 1458. The Hunyadi family had unknown origin. Moreover, Matthias failed to found a dynasty because of lacking a legitimate heir and his illegitimate son Johannes Corvinus was unable to obtain the crown. His grandson, Christophorus Corvinus, died in childhood, thus the direct male line of the family ended. In the framework of on interdisciplinary research, we have determined the whole genome sequences of Johannes Corvinus and Christophorus Corvinus by next-generation sequencing technology. Both of them carried the Y-chromosome haplogroup is E1b1b1a1b1a6a1c ∼, which is widespread in Eurasia. The father-son relationship was verified using the classical STR method and whole genome data. Christophorus Corvinus belongs to the rare, sporadically occurring T2c1+146 mitochondrial haplogroup, most frequent around the Mediterranean, while his father belongs to the T2b mitochondrial haplogroup, widespread in Eurasia, both are consistent with the known origin of the mothers. Archaeogenomic analysis indicated that the Corvinus had an ancient European genome composition. Based on the reported genetic data, it will be possible to identify all the other Hunyadi family member, whose only known grave site is known, but who are resting assorted with several other skeletons.

Revealing a Phenotypical Appearance of Ibrutinib Resistance in Patients With Chronic Lymphocytic Leukaemia by Flow Cytometry.

Background: Ibrutinib is widely known as an effective and well-tolerated therapeutical choice of the chronic lymphocytic leukaemia (CLL). However, acquired resistance may occur during the treatment, causing relapse. Early detection of ibrutinib resistance is an important issue, therefore we aimed to find phenotypic markers on CLL cells the expression of which may correlate with the appearance of ibrutinib resistance. Methods: We examined 28 patients' peripheral blood (PB) samples (treatment naïve, ibrutinib sensitive, clinically ibrutinib resistant). The surface markers' expression (CD27, CD69, CD86, CD184, CD185) were measured by flow cytometry. Furthermore, the BTKC481S resistance mutation was assessed by digital droplet PCR. Moreover, the CLL cells' phenotype of a patient with acquired ibrutinib resistance was observed during the ibrutinib treatment. Results: The expression of CD27 (p = 0.030) and CD86 (p = 0.031) became higher in the clinically resistant cohort than in the ibrutinib sensitive cohort. Besides, we found that high CD86 and CD27 expressions were accompanied by BTKC481S mutation. Our prospective study showed that the increase of the expression of CD27, CD69 and CD86 was noticed ahead of the clinical resistance with 3 months. Conclusion: Our study suggests that the changes of the expression of these markers could indicate ibrutinib resistance and the examination of these phenotypic changes may become a part of the patients' follow-up in the future.

Screening and monitoring of the BTKC481S mutation in a real-world cohort of patients with relapsed/refractory chronic lymphocytic leukaemia during ibrutinib therapy.

The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib has revolutionised the therapeutic landscape of chronic lymphocytic leukaemia (CLL). Acquired mutations emerging at position C481 in the BTK tyrosine kinase domain are the predominant genetic alterations associated with secondary ibrutinib resistance. To assess the correlation between disease progression, and the emergence and temporal dynamics of the most common resistance mutation BTKC481S , sensitive (10-4 ) time-resolved screening was performed in 83 relapsed/refractory CLL patients during single-agent ibrutinib treatment. With a median follow-up time of 40 months, BTKC481S was detected in 48·2% (40/83) of the patients, with 80·0% (32/40) of them showing disease progression during the examined period. In these 32 cases, representing 72·7% (32/44) of all patients experiencing relapse, emergence of the BTKC481S mutation preceded the symptoms of clinical relapse with a median of nine months. Subsequent Bcl-2 inhibition therapy applied in 28/32 patients harbouring BTKC481S and progressing on ibrutinib conferred clinical and molecular remission across the patients. Our study demonstrates the clinical value of sensitive BTKC481S monitoring with the largest longitudinally analysed real-world patient cohort reported to date and validates the feasibility of an early prediction of relapse in the majority of ibrutinib-treated relapsed/refractory CLL patients experiencing disease progression.

Surgery formulae for the Seiberg-Witten invariant of plumbed 3-manifolds.

Assume that M ( T ) is a rational homology sphere plumbed 3-manifold associated with a connected negative definite graph T . We consider the combinatorial multivariable Poincaré series associated with T and its counting functions, which encode rich topological information. Using the 'periodic constant' of the series (with reduced variables associated with an arbitrary subset I of the set of vertices) we prove surgery formulae for the normalized Seiberg-Witten invariants: the periodic constant associated with I appears as the difference of the Seiberg-Witten invariants of M ( T ) and M ( T \ I ) for any I .

In situ cell cycle analysis in giant cell tumor of bone reveals patients with elevated risk of reduced progression-free survival.

OBJECTIVE: Giant cell tumor of bone (GCTB) is a frequently recurring locally aggressive osteolytic lesion, where pathological osteoclastogenesis and bone destruction are driven by neoplastic stromal cells. Here, we studied if cell cycle fractions within the mononuclear cell compartment of GCTB can predict its progression-free survival (PFS). METHODS: 154 cases (100 primaries and 54 recurrent) from 139 patients of 40 progression events, was studied using tissue microarrays. Ploidy and in situ cell cycle progression related proteins including Ki67 and those linked with replication licensing (mcm2), G1-phase (cyclin D1, Cdk4), and S-G2-M-phase (cyclin A; Cdk2) fractions; cell cycle control (p21waf1) and repression (geminin), were tested. The Prentice-Williams-Peterson (PWP) gap-time models with the Akaike information criterion (AIC) were used for PFS analysis. RESULTS: Cluster analysis showed good correlation between functionally related marker positive cell fractions indicating no major cell cycle arrested cell populations in GCTB. Increasing hazard of progression was statistically associated with the elevated post-G1/S-phase cell fractions. Univariate analysis revealed significant negative association of poly-/aneuploidy (p < 0.0001), and elevated cyclin A (p < 0.001), geminin (p = 0.015), mcm2 (p = 0.016), cyclin D1 (p = 0.022) and Ki67 (B56: p = 0.0543; and Mib1: p = 0.0564 -strong trend) positive cell fractions with PFS. The highest-ranked multivariate interaction model (AIC = 269.5) also included ploidy (HR 5.68, 95%CI: 2.62-12.31, p < 0.0001), mcm2 (p = 0.609), cyclin D1 (HR 1.89, 95%CI: 0.88-4.09, p = 0.105) and cyclin A (p < 0.0001). The first and second best prognostic models without interaction (AIC = 271.6) and the sensitivity analysis (AIC = 265.7) further confirmed the prognostic relevance of combining these markers. CONCLUSION: Ploidy and elevated replication licensing (mcm2), G1-phase (cyclin D1) and post-G1 phase (cyclin A) marker positive cell fractions, indicating enhanced cell cycle progression, can assist in identifying GCTB patients with increased risk for a reduced PFS.

Endophytic fungi from the roots of horseradish (Armoracia rusticana) and their interactions with the defensive metabolites of the glucosinolate - myrosinase - isothiocyanate system.

BACKGROUND: The health of plants is heavily influenced by the intensively researched plant microbiome. The microbiome has to cope with the plant's defensive secondary metabolites to survive and develop, but studies that describe this interaction are rare. In the current study, we describe interactions of endophytic fungi with a widely researched chemical defense system, the glucosinolate - myrosinase - isothiocyanate system. The antifungal isothiocyanates are also of special interest because of their beneficial effects on human consumers. RESULTS: Seven endophytic fungi were isolated from horseradish roots (Armoracia rusticana), from the genera Fusarium, Macrophomina, Setophoma, Paraphoma and Oidiodendron. LC-ESI-MS analysis of the horseradish extract incubated with these fungi showed that six of seven strains could decompose different classes of glucosinolates. Aliphatic, aromatic, thiomethylalkyl and indolic glucosinolates were decomposed by different strains at different rates. SPME-GC-MS measurements showed that two strains released significant amounts of allyl isothiocyanate into the surrounding air, but allyl nitrile was not detected. The LC-ESI-MS analysis of many strains' media showed the presence of allyl isothiocyanate - glutathione conjugate during the decomposition of sinigrin. Four endophytic strains also accepted sinigrin as the sole carbon source. Isothiocyanates inhibited the growth of fungi at various concentrations, phenylethyl isothiocyanate was more potent than allyl isothiocyanate (mean IC50 was 2.30-fold lower). As a control group, ten soil fungi from the same soil were used. They decomposed glucosinolates with lower overall efficiency: six of ten strains had insignificant or weak activities and only three could use sinigrin as a carbon source. The soil fungi also showed lower AITC tolerance in the growth inhibition assay: the median IC50 values were 0.1925 mM for endophytes and 0.0899 mM for soil fungi. CONCLUSIONS: The host's glucosinolates can be used by the tested endophytic fungi as nutrients or to gain competitive advantage over less tolerant species. These activities were much less apparent among the soil fungi. This suggests that the endophytes show adaptation to the host plant's secondary metabolites and that host metabolite specific activities are enriched in the root microbiome. The results present background mechanisms enabling an understanding of how plants shape their microbiome.

Effect of pH on stability and plasmonic properties of cysteine-functionalized silver nanoparticle dispersion.

Citrate-stabilized spherical silver nanoparticles (Ag NPs) with d=8.25±1.25 nm diameter were prepared and functionalized with L-cysteine (Cys) in aqueous dispersion. The nanosilver-cysteine interactions have been investigated by Raman and (1)H NMR spectroscopy. The effect of pH on stability of biofunctionalized Ag NPs was investigated. The cysteine-capped nanosilver dispersions remain stable at higher pH (pH>7), while the degree of aggregation increased as the pH decreased. Below pH ~7, the characteristic surface plasmon band of bare silver nanoparticles was back-shifted from λ(measured)(bareAgNP)=391 nm to λ(measured)(1)=387-391 nm, while the presence of a new band at λ(measured)(2)=550-600 nm was also observed depending on pH. Finite element method (FEM) was applied to numerically compute the absorption spectra of aqueous dispersions containing bare and cysteine-functionalized Ag NPs at different pH. Both the dynamic light scattering (DLS) measurements, Zeta potential values and the transmission electron microscopic (TEM) images confirmed our supposition. Namely, electrostatic interaction arose between the deprotonated carboxylate (COO(-)) and protonated amino groups (NH(3)(+)) of the amino acid resulting in cross-linking network of the Ag NPs between pH ~3 and 7. If the pH is measurable lower than ~3, parallel with the protonation of citrate and L-cysteine molecules the connection of the particles via l-cysteine is partly decomposed resulting in decrease of second plasmon band intensity.