A New Heart-Cutting Method for a Multiplex Quantitative Analysis of Steroid Hormones in Plasma Using 2D-LC/MS/MS Technique.

The aim of the current research was to develop a simple and rapid mass spectrometry-based assay for the determination of 15 steroid hormones in human plasma in a single run, which would be suitable for a routine practice setting. For this purpose, we designed a procedure based on the 2D-liquid chromatography-tandem mass spectrometry with a minimalistic sample pre-treatment. In our arrangement, the preparation of one sample takes only 10 min and can accommodate 40 samples per hour when tested in series. The following analytical run is 18 min long for all steroid hormones. In addition, we developed an independent analytical run for estradiol, significantly increasing the assay accuracy while taking an additional 10 min to perform an analytical run of a sample. The optimized method was applied to a set of human plasma samples, including chylous. Our results indicate the linearity of the method for all steroid hormones with squared regression coefficients R2 ≥ 0.995, within-run and between-run precision (RSD < 6.4%), and an accuracy of 92.9% to 106.2%. The absolute recovery for each analyzed steroid hormone ranged between 101.6% and 116.5%. The method detection limit for 15 steroid hormones ranged between 0.008 nmol/L (2.88 pg/mL) for aldosterone and 0.873 nmol/L (0.252 ng/mL) for DHEA. For all the analytes, the lowest calibration point relative standard deviation was less than 10.8%, indicating a good precision of the assay within the lowest concentration of interest. In conclusion, in this method article, we describe a simple, sensitive, and cost-effective 2D-LC/MS/MS method suitable for the routine analysis of a complex of steroid hormones allowing high analytical specificity and sensitivity despite minimal sample processing and short throughput times.

Rapid genome sequencing for pediatrics.

The advancements made in next-generation sequencing (NGS) technology over the past two decades have transformed our understanding of genetic variation in humans and had a profound impact on our ability to diagnose patients with rare genetic diseases. In this review, we discuss the recently developed application of rapid NGS techniques, used to diagnose pediatric patients with suspected rare diseases who are critically ill. We highlight the challenges associated with performing such clinical diagnostics tests in terms of the laboratory infrastructure, bioinformatic analysis pipelines, and the ethical considerations that need to be addressed. We end by looking at what future developments in this field may look like and how they can be used to augment the genetic data to further improve the diagnostic rates for these high-priority patients.

The winner takes it all: dominance of Calicophoron daubneyi (Digenea: Paramphistomidae) among flukes in Central European beef cattle.

In Europe, paramphistomosis caused by Paramphistomum spp. was historically regarded as being of minor importance. However, Calicophoron daubneyi has recently been recognized as an emerging pathogen in Europe due to its increasing prevalence and negative impact on livestock production. In search for paramphistomid flukes, 5573 beef cattle fecal samples from 115 farms across the whole Czech Republic were examined from March 2019 to June 2021. The eggs of paramphistomid flukes were identified in 29.9% of samples. Internal transcribed spacer 2 sequences from 90 adult flukes and 125 fecal samples collected across Czech Republic confirmed C. daubneyi infection in the Czech beef cattle. Ninety mitochondrial DNA sequences obtained from adult C. daubneyi specimens revealed 13 individual haplotypes, two of them recorded for the first time. Although C. daubneyi is a new parasite in beef cattle herds in the Czechia, it clearly dominates the parasitological findings in the country's beef cattle. The common occurrence of C. daubneyi in most of the beef cattle herds indicates environmental conditions suitable also for the life cycle of Fasciola hepatica and risk of its emergence.

Cryptosporidium ratti n. sp. (Apicomplexa: Cryptosporidiidae) and genetic diversity of Cryptosporidium spp. in brown rats (Rattus norvegicus) in the Czech Republic.

The diversity and biology of Cryptosporidium that is specific for rats (Rattus spp.) are not well studied. We examined the occurrence and genetic diversity of Cryptosporidium spp. in wild brown rats (Rattus norvegicus) by microscopy and polymerase chain reaction (PCR)/sequencing targeting the small subunit rDNA (SSU), actin and HSP70 genes. Out of 343 faecal samples tested, none were positive by microscopy and 55 were positive by PCR. Sequence analysis of SSU gene revealed the presence of Cryptosporidium muris (n = 4), C. andersoni (n = 3), C. ryanae (n = 1), C. occultus (n = 3), Cryptosporidium rat genotype I (n = 23), Cryptosporidium rat genotype IV (n = 16) and novel Cryptosporidium rat genotype V (n = 5). Spherical oocysts of Cryptosporidium rat genotype I obtained from naturally-infected rats, measuring 4.4-5.4 µm × 4.3-5.1 µm, were infectious to the laboratory rats, but not to the BALB/c mice (Mus musculus) nor Mongolian gerbils (Meriones unguiculatus). The prepatent period was 3 days post infection and the patent period was longer than 30 days. Naturally- and experimentally-infected rats showed no clinical signs of disease. Percentage of nucleotide similarities at the SSU, actin, HSP70 loci between C. ratti n. sp. and the rat derived C. occultus and Cryptosporidium rat genotype II, III, IV, and V ranged from 91.0 to 98.1%. These genetic variations were similar or greater than that observed between closely related species, i.e. C. parvum and C. erinacei (93.2-99.5%). Our morphological, genetic and biological data support the establishment of Cryptosporidium rat genotype I as a new species, Cryptosporidium ratti n. sp.

Pharmacologically induced thyreopathy in patients with cardiovascular disease.

Thyroid gland function is mediated by thyreoideal hormones, in which iodine is very important structural part. High iodine intake, can initiate thyroid dysfunction. Amiodarone induced hypothyroidism is treated with levothyroxine and amiodarone taking is not interrupted. Amiodarone induced hyperthyroidism is divided into two subtypes, which differ by mechanism of origin and treatment strategy. In patients with cardiovascular disease is higher possibility of getting substances, with high content of iodine in diagnostic-therapeutic examination with contrast or treatment with amiodarone. In this group of patients is necessary to control thyroid function regularly and to hold preventive actions.

Role of gamma knife radiosurgery in the treatment of prolactinomas.

PURPOSE: Stereotactic radiosurgery is one of the treatment options for prolactinomas, the most commonly used being Gamma Knife Radiosurgery (GKRS). GKRS is indicated mainly in the treatment of dopamine agonist (DA)-resistant prolactinomas. In our study, we report on our experience in treating prolactinoma patients by GKRS. METHODS: Twenty-eight patients were followed-up after GKRS for 26-195 months (median 140 months). Prior to GKRS, patients were treated with DAs and 9 of them (32.1%) underwent previous neurosurgery. Cavernous sinus invasion was present in 16 (57.1%) patients. Indications for GKRS were (i) resistance to DA treatment (17 patients), (ii) drug intolerance (5 patients), or (iii) attempts to reduce the dosage and/or shorten the length of DA treatment (6 patients). RESULTS: After GKRS, normoprolactinaemia was achieved in 82.1% of patients, out of which hormonal remission (normoprolactinaemia after discontinuation of DAs) was achieved in 13 (46.4%), and hormonal control (normoprolactinaemia while taking DAs) in 10 (35.7%) patients. GKRS arrested adenoma growth or decreased adenoma size in all cases. Two patients (8.3%) developed hypopituitarism after GKRS. Prolactinoma cystic transformation with expansive behaviour, manifested by bilateral hemianopsia, was observed in one patient. CONCLUSIONS: GKRS represents an effective treatment option, particularly for DA-resistant prolactinomas. Normoprolactinaemia was achieved in the majority of patients, either after discontinuation of, or while continuing to take, DAs. Tumour growth was arrested in all cases. The risk of the development of hypopituitarism can be limited if the safe dose to the pituitary and infundibulum is maintained.

Human germ/stem cell-specific gene TEX19 influences cancer cell proliferation and cancer prognosis.

BACKGROUND: Cancer/testis (CT) genes have expression normally restricted to the testis, but become activated during oncogenesis, so they have excellent potential as cancer-specific biomarkers. Evidence is starting to emerge to indicate that they also provide function(s) in the oncogenic programme. Human TEX19 is a recently identified CT gene, but a functional role for TEX19 in cancer has not yet been defined. METHODS: siRNA was used to deplete TEX19 levels in various cancer cell lines. This was extended using shRNA to deplete TEX19 in vivo. Western blotting, fluorescence activated cell sorting and immunofluorescence were used to study the effect of TEX19 depletion in cancer cells and to localize TEX19 in normal testis and cancer cells/tissues. RT-qPCR and RNA sequencing were employed to determine the changes to the transcriptome of cancer cells depleted for TEX19 and Kaplan-Meier plots were generated to explore the relationship between TEX19 expression and prognosis for a range of cancer types. RESULTS: Depletion of TEX19 levels in a range of cancer cell lines in vitro and in vivo restricts cellular proliferation/self-renewal/reduces tumour volume, indicating TEX19 is required for cancer cell proliferative/self-renewal potential. Analysis of cells depleted for TEX19 indicates they enter a quiescent-like state and have subtle defects in S-phase progression. TEX19 is present in both the nucleus and cytoplasm in both cancerous cells and normal testis. In cancer cells, localization switches in a context-dependent fashion. Transcriptome analysis of TEX19 depleted cells reveals altered transcript levels of a number of cancer-/proliferation-associated genes, suggesting that TEX19 could control oncogenic proliferation via a transcript/transcription regulation pathway. Finally, overall survival analysis of high verses low TEX19 expressing tumours indicates that TEX19 expression is linked to prognostic outcomes in different tumour types. CONCLUSIONS: TEX19 is required to drive cell proliferation in a range of cancer cell types, possibly mediated via an oncogenic transcript regulation mechanism. TEX19 expression is linked to a poor prognosis for some cancers and collectively these findings indicate that not only can TEX19 expression serve as a novel cancer biomarker, but may also offer a cancer-specific therapeutic target with broad spectrum potential.

[Treatment of pituitary adenomas]. / Lécba hypofyzárních adenomu.

Pituitary adenomas are the most common tumours of the sellar region. A combination of neurosurgery, radiation and pharmacological approaches are applied for the treatment of pituitary adenomas. In certain cases, patient observation is another option. Neurosurgery is the first-choice treatment for acromegaly, Cushing´s disease and TSH secreting adenomas. Leksell gamma knife irradiation is used in the treatment of tumour residues. Until the effect of the irradiation is evident, pharmacological treatment must be administered. Large and/or growing non-functioning pituitary adenomas are operated. Irradiation is possible if there is sufficient distance between the margin of the adenoma and the optic pathway. The primary therapy for prolactinomas is pharmacological treatment with dopamine agonists. Multidisciplinary collaboration among endocrinologists, neurosurgeons and radiosurgeons is necessary in the treatment of pituitary adenomas.

Cryptosporidium testudinis sp. n., Cryptosporidium ducismarci Traversa, 2010 and Cryptosporidium tortoise genotype III (Apicomplexa: Cryptosporidiidae) in tortoises.

Understanding of the diversity of species of Cryptosporidium Tyzzer, 1910 in tortoises remains incomplete due to the limited number of studies on these hosts. The aim of the present study was to characterise the genetic diversity and biology of cryptosporidia in tortoises of the family Testudinidae Batsch. Faecal samples were individually collected immediately after defecation and were screened for presence of cryptosporidia by microscopy using aniline-carbol-methyl violet staining, and by PCR amplification and sequence analysis targeting the small subunit rRNA (SSU), Cryptosporidium oocyst wall protein (COWP) and actin genes. Out of 387 faecal samples from 16 tortoise species belonging to 11 genera, 10 and 46 were positive for cryptosporidia by microscopy and PCR, respectively. All samples positive by microscopy were also PCR positive. Sequence analysis of amplified genes revealed the presence of the Cryptosporidium tortoise genotype I (n = 22), C. ducismarci Traversa, 2010 (n = 23) and tortoise genotype III (n = 1). Phylogenetic analyses of SSU, COWP and actin gene sequences revealed that Cryptosporidium tortoise genotype I and C. ducismarci are genetically distinct from previously described species of Cryptosporidium. Oocysts of Cryptosporidium tortoise genotype I, measuring 5.8-6.9 µm × 5.3-6.5 µm, are morphologically distinguishable from C. ducismarci, measuring 4.4-5.4 µm × 4.3-5.3 µm. Oocysts of Cryptosporidium tortoise genotype I and C. ducismarci obtained from naturally infected Russian tortoises (Testudo horsfieldii Gray) were infectious for the same tortoise but not for Reeve's turtles (Mauremys reevesii [Gray]), common garter snake (Thamnophis sirtalis [Linnaeus]), zebra finches (Taeniopygia guttata [Vieillot]) and SCID mice (Mus musculus Linnaeus). The prepatent period was 11 and 6 days post infection (DPI) for Cryptosporidium tortoise genotype I and C. ducismarci, respectively; the patent period was longer than 200 days for both cryptosporidia. Naturally or experimentally infected tortoises showed no clinical signs of disease. Our morphological, genetic, and biological data support the establishment of Cryptosporidium tortoise genotype I as a new species, Cryptosporidium testudinis sp. n., and confirm the validity of C. ducismarci as a separate species of the genus Cryptosporidium.

[TSH secreting adenomas]. / TSH produkující adenomy.

Adenomas which secrete thyrotropic hormone (thyrotropinomas) are rare and constitute less than 3 % of pituitary adenomas. In laboratory studies there is a typical elevation of thyroid hormones with nonsupressible TSH. In differential diagnostics it is necessary to distinguish above all the syndrome of resistance to thyroid hormones. Clinical symptoms are usually mild and correspond to symptoms of hyperthyroidism. Goiter is a common finding. In 80 % of cases thyrotropinomas are diagnosed in a stage of invasively growing macroadenoma. The primary treatment is neurosurgical removal adenoma which results in cure in 40 % of patients. Other treatment options include radiation therapy and medical treatment (treatment with somatostatin analogues). With regard to the risk of adenoma recurrence, the long-term follow-up is similar to that of cases of other pituitary adenomas necessary.Key words: resistance to thyroid hormone - TSH secreting adenoma - thyreotropin.