Low back pain in patients hospitalised with liver cirrhosis- a retrospective study.

BACKGROUND: Lower back pain is a common issue, but little is known about the prevalence of pain in patients with liver cirrhosis during hospitalisation. Therefore, the objective of this study was to determine lower back pain in patients with liver cirrhosis. METHODS: The sample consisted of patients with liver cirrhosis (n = 79; men n = 55; women n = 24; mean age = 55.79 ± 12.52 years). The hospitalised patients were mobile. The presence and intensity of pain were assessed in the lumbar spine during hospitalisation. The presence of pain was assessed using the visual analogue pain scale (0-10). The range of motion of the lower spine was assessed using the Schober and Stibor tests. Frailty was measured by Liver Frailty Index (LFI). The condition of liver disease was evaluated using The Model For the End-Stage Liver Disease (MELD) and Child-Pugh score (CPS) and ascites classification. Student's t test and Mann-Whitney test were used for analysis of the difference of group. Analysis of variance (ANOVA) with the Tukey post hoc test was used to test differences between categories of liver frailty index. The Kruskal-Wallis test was used to test pain distribution. Statistical significance was determined at the α-0.05 significance level. RESULT: The prevalence of pain in patients with liver cirrhosis was 13.92% (n = 11), and the mean intensity of pain according to the visual analogue scale was 3.73 (± 1.90). Lower back pain was present in patients with ascites (15.91%; n = 7) and without ascites (11.43%; n = 4). The prevalence of lower back pain was not statistically significant between patients with and without ascites (p = 0,426). The base of Schober's assessment mean score was 3.74 cm (± 1.81), and based on Stibor's assessment mean score was 5.84 cm (± 2.23). CONCLUSION: Lower back pain in patients with liver cirrhosis is a problem that requires attention. Restricted spinal mobility has been reported in patients with back pain, according to Stibor, compared to patients without pain. There was no difference in the incidence of pain in patients with and without ascites.

Rehabilitation and physical activity for COVID-19 patients in the post infection period.

The first cases of COVID-19 were initially recorded in December 2019 in Wuhan, the capital of China's Hubei Province. The situation quickly escalated and turned into a global pandemic. COVID-19 is a highly infectious respiratory disease that leads to decreased respiratory, physical, and psychological function of affected patients (2). Patients' symptoms widely vary; from asymptomatic course to severe symptoms. Decrease in physical function, and, in some cases, a persistence of symptoms may be observed in patients, who overcame the infection period. Rehabilitation represents a potential treatment option for COVID-19 patients in post-infection period. Rehabilitation therapies may help to restore physical function in patients and to reduce the long-term effects of COVID19 infection (Ref. 37). Text in PDF www.elis.sk Keywords: COVID19, rehabilitation, post-infection syndrome.

Small bowel adenocarcinoma in Crohn's disease: a rare but devastating complication.

BACKGROUND: Small bowel adenocarcinoma (SBA) remains a rare entity but occurs at increased frequency in the setting of chronic Crohn's disease (CD). Our aim was to study the presentation, diagnosis and prognosis of SBA in patients undergoing surgery for CD at a single institution. METHODS: We reviewed the medical records of all patients with CD complicated by adenocarcinoma of the small bowel from 2000 to 2017. Descriptive statistics and Kaplan-Meier overall survival estimates were calculated. RESULTS: In total, 22 patients (14 males) with CD (median duration of Crohn's diagnosis 32 years) were diagnosed with SBA and underwent surgical resection (8 isolated small bowel resections, 12 ileocolic resections, and 2 total proctocolectomies). The median patient age at the time of diagnosis was 54 years (range 22-82 years). A total of 17 patients (77%) underwent cross-sectional CT imaging within 3 months of surgery, a cancer diagnosis was suggested in only one patient. In one other patient, SBA was diagnosed preoperatively on endoscopic biopsy of the terminal ileum. The remaining patients were operated on for obstruction (n = 17), abscess or fistulizing disease (n = 2), and sigmoid cancer (n = 1). For these 20 (90%) patients not suspected to have SBA on preoperative assessment, 5 (25%) were diagnosed intraoperatively on frozen section and 15 (75%) were unexpectedly diagnosed postoperatively on final pathology. T staging was characterized by more advanced tumors (T4: 59%, T3: 27%, T2: 9%, and T1: 5%). Nine patients (41%) had nodal involvement and five patients (23%) had hepatic and/or peritoneal carcinomatosis. The 1-, 3-, and 5-year survival estimates for our cohort were 84%, 30%, and 10%, respectively. Median survival was 30.5 months with median follow-up of 23 months (range 6-84 months). CONCLUSIONS: SBA in the setting of CD is most commonly found incidentally after surgical resection for benign indications. As such, any suspicious finding at the time of surgery in a patient with chronic CD should warrant careful investigation with frozen section and/or resection. Prognosis for CD complicated by SBA remains poor even in the modern era.

Conservative treatment options for polycystic ovary syndrome: the importance of exercise.

AIM: The main aim of the article is inform about benefits of rehabilitation in the treatment of polycystic ovary syndrome (PCOS). STUDY TYPE: Review article. METHODS: Search for meta-analyzes and systematic review in Pubmed, Medline, Scopus databases. RESULTS: Insulin resistance plays a potential role in the pathogenesis of PCOS. Lifestyle modification (including physical activity and diet) may help alleviate the symptoms of PCOS, as lifestyle factors may reduce insulin resistance and improve metabolism and reproductive function. The benefit of exercise in women with PCOS is also associated with other significant benefits such as improved anxiety, metabolic syndrome and improved regularity of the menstrual cycle. CONCLUSION: Rehabilitation is an important part of therapy in patients with PCOS. Aerobic, strength and interval exercises has a significant benefit in the treatment of polycystic ovary syndrome.

Benefits of exercise in the prenatal and postnatal period.

AIM: The main aim of the article is find benefits of exercise during and after pregnancy. STUDY TYPE: Review article. METHODS: Search for meta-analyzes and system reports in PubMed, JCR, Medline, Scopus databases. RESULT: Regular exercise is associated with a lower risk of macrosomy and caesarean section during childbirth. Depression is a  common complication in the prenatal and postnatal period due to increased stress and insufficient social support. Exercise is a potential non-pharmacological therapy. Regular exercise is also associated with lowering blood glucose and improving insulin sensitivity. To optimize weight and overweight, exercise is one of the main parts of treatment. Another therapeutic possibility of using the exercise is to influence the pain in the lumbosacral area and also the pain in the pelvic area. Another variant of exercises are exercises to strengthen the pelvic floor. Exercise of the pelvic floor muscles in the perinatal period is an effective method for preventing postpartum urinary incontinence. CONCLUSION: Exercise is an important part of therapy in the prenatal and postnatal period.

[Clinical Assessment of Anterior Cruciate Ligament Rupture]. / Ruptúra predného krízneho väzu a manuálne vysetrenie.

Anterior cruciate ligament (ACL) rupture is one of the most common traumatic injuries of the knee joint. Acute knee injury is often characterized by pain and the typical accompanying rupture sound. The injured person often feels pain in the knee, with swelling, the movement is painful in the full range of motion. The most commonly used test procedures for rupture include Lachman test, pivot shift test, anterior drawer and lever sign test. This review includes a description of individual tests and the diagnostic value of examination after the ACL rupture. The sensitivity and specificity of the lever sign test was 0.92-1.00, the specificity was 0.94-1.00. The anterior drawer testing reported sensitivity values ranged from 0.18 to 0.92 and specificity values ranged from 0.78 to 0.98. The sensitivity and specificity of the pivot shift test ranged from 0.18 to 0.48 and the specificity from 0.90 to 0.99. The sensitivity and specificity of the Lachman test were 0.63-0.93 and the specificity was 0.55-0.99. The lever sign test, the pivot shift test, the anterior drawer test and the Lachman test are valid parts of the anterior cruciate ligament examination with respect to the prediction of anterior cruciate ligament rupture using Magnetic Resonance Imaging and arthroscopy. Key words: rupture ligamentum cruciatum anterius, test maneuvers, lever sign test, pivot shift test, anterior drawer, Lachman test.

The relationship between mesorectal grading and oncological outcome in rectal adenocarcinoma.

AIM: The prognostic association between mesorectal grading and oncological outcome in patients undergoing resection for rectal adenocarcinoma is controversial. The aim of this retrospective chart review was to determine the individual impact of mesorectal grading on rectal cancer outcomes. METHOD: We compared oncological outcomes in patients with complete, near-complete and incomplete mesorectum who underwent rectal excision with curative intent from 2009 to 2014 for Stage cI-III rectal adenocarcinoma. We also assessed the independent association of mesorectal grading and oncological outcome using multivariate models including other relevant variables. RESULTS: Out of 505 patients (339 men, median age of 60 years), 347 (69%) underwent a restorative procedure. There were 452 (89.5%), 33 (6.5%) and 20 (4%) patients with a complete, near-complete and incomplete mesorectum, respectively. Local recurrence was seen in 2.4% (n = 12) patients after a mean follow-up of 3.1 ± 1.7 years. Unadjusted 3-year Kaplan-Meier analysis by mesorectal grade showed decreased rates of overall, disease-free and cancer-specific survival and increased rates of overall and distant recurrence with a near-complete mesorectum, while local recurrence was increased in cases of an incomplete mesorectum (all P < 0.05). On multivariate analyses, a near-complete mesorectum was independently associated with decreased cancer-specific survival (hazard ratio 0.26, 95% CI 0.1-0.7; P = 0.007). There were no associations between mesorectal grading and overall survival, disease-free survival, overall recurrence or distant recurrence (all P > 0.05). CONCLUSION: Mesorectal grading is independently associated with oncological outcome. It provides unique information for optimizing surgical quality in rectal cancer.

Outcomes of ileal pouch-anal anastomosis without primary diverting loop ileostomy if postoperative sepsis develops.

BACKGROUND: The aim of the present study was to assess the short-term and long-term consequences of diverting loop ileostomy (DLI) omission in ileal pouch-anal anastomosis (IPAA) surgery complicated by postoperative pelvic sepsis. METHODS: This was a retrospective review of a prospectively maintained database. Of 4031 patients who underwent IPAA in 1983-2014, 357 developed IPAA-related pelvic sepsis with or without anastomotic dehiscence. Patients with Crohn's disease or cancer were excluded. The patient cohort was divided into two groups, depending on the presence or absence of DLI. Patient characteristics, short-term and long-term outcomes were compared. Long-term pouch survival was estimated with the Kaplan-Meier method. Quality of life (QOL) in the groups was compared at the latest follow-up. RESULTS: Three hundred and twenty-six patients developing pelvic sepsis had diversion at the time of IPAA (D group) and in 31 who developed pelvic sepsis DLI had been omitted (O group). The length of hospital stay was significantly longer in the O group 11.5 (3-33) days versus 8 (2-59) days in the D group (p = 0.006). Forty-eight percent of patients from the O group with anastomotic leak underwent reoperation and had a DLI formed at this second procedure versus 12% in the D group requiring reoperation (p < 0.0001). In long-term follow-up, there was no difference in pouch survival between the groups: 99 versus 97% after 5 years and 88 versus 87% after 10 years, in the O group and D group, respectively (p = 0.40). There was no difference in QOL observed between the groups. CONCLUSIONS: Omission of DLI in selected patients who had IPAA surgery did not increase pouch failure or adversely affect QOL in the long term, if pelvic sepsis occurred.

Comparison of the level of physical activity after the COVID-19 pandemic in Poland, Slovakia and the Czech Republic.

BACKGROUND: The COVID-19 pandemic was associated with limited physical activity (PA) of most of the world's population. This cross-sectional prospective study aimed to assess the levels of PA of university students in Poland, Czech Republic and Slovakia after COVID-19 using the International Physical Activity Questionnaire Short Form (IPAQ-SF). METHODS: A total of 2635 students completed questionnaires regarding their PA levels using the IPAQ-SF between September and December 2022. RESULTS: PA measured by metabolic equivalent of task (MET) scores, varied between the three countries: Slovakia median MET-minutes/week score 4459.9; Czech Republic 3838.8 Poland 3567.1. The results of the post hoc analysis revealed there were significant differences in MET-minutes/week values between the Czech Republic and Poland (p < 0.035) as well as between the Czech Republic and Slovakia (p < 0.037). The analysis of energetic expenditure during walking revealed that students from the Czech Republic and Slovakia had higher median MET-min/weeks values (Czech 2284.1; Slovak 2467.1) compared to their Polish (1536.1) peers (p < 0.001). Polish cohort presented with significantly higher body mass index (BMI) (p < 0.001) than Czech and Slovak groups (BMI Czech: 22.3; Slovak 22.8; Polish 23.8). CONCLUSIONS: Significant differences in PA levels between the Czech Republic, Poland, and Slovakia university students were identified. Slovakia showed the highest median PA measured as a MET score, and Poland showed the lowest. Compared to available pre-COVID studies it seems the total level of PA in the observed cohorts has not returned to the pre-COVID levels and students remain less active.

Comparison of the quality of life of patients with liver cirrhosis before and during the COVID-19 lockdown in Slovakia.

Liver cirrhosis is associated with a poor quality of life (QOL). The COVID-19 pandemic has led to several restriction measures and psychosocial consequences whose impact on QOL has combined with that of cirrhosis in an unknown way. Therefore, we have used our cirrhosis registry to assess the quality of life before the pandemic (on the first admission to the tertiary liver unit) and during the most pronounced phase of the first lockdown. In this cross-sectional study conducted during the first lockdown in Slovakia (from April to May 2020), we have repeated the QOL measurement of QOL in cirrhotic patients previously enrolled in the RH7 registry. Patients who were alive (according to the national registry of deaths) were identified and contacted by phone with a structured and standardized interview led by trained professionals. The tool used for both QOL measurements (at enrolment in RH7 and during lockdown) was a standardized and validated EuroQOL-5D (EQ-5D) questionnaire. The study included 97 patients, of which 37 (38.1%) were women and 60 (61.9%) were men. Responses were achieved from 75 patients (68.18%). In general, patients scored their quality of life significantly higher during the pandemic compared to examination at admission to RH7 (that is, at admission to our tertiary liver unit with cirrhosis) (p = 0.005). In particular, of the domains included in EQ-5D: (1) self-care was better during lockdown compared to the first record on admission to RH7 (p < 0.001). (2) the ability to perform daily activities has also improved during lockdown (p = 0.002). On the other hand, (3) pain and discomfort did not change significantly during the lockdown compared to the previous measurement (p = 0.882). (4) anxiety and depression were lower during lockdown compared to admission to RH7 (p = 0.01). The quality of life in patients with liver cirrhosis was better during the lockdown of SARS-CoV-2 compared to the previous measurement at admission to the tertiary liver unit.

Rehabilitation and physical activity in gynecological oncological diseases.

BACKGROUND: The cause of gynecological tumors is multifactorial. Risk factors include higher BMI and lack of physical activity. Gynecological oncological diseases are associated with loss of function due to the pathophysiological effects of the disease, treatment, and also mental stress in patients. These problems lead to greater rehabilitation demand of patients. Rehabilitation aims to help the patient to achieve the best possible level of functional status, to foster independence, to improve acceptance of the disease, to improve patient fatigue and improve the quality of life of the patients It is essential to focus the rehabilitation examination on several factors associated with impaired function, such as impaired cardiovascular and pulmonary function, urinary incontinence or psychological or psychological distress. The pleiotropic effect of rehabilitation can also be used in pain relief, improvement of chemotherapy tolerance, in the treatment of lymphedema and in the improvement of pelvic floor muscle function. PURPOSE: The main aim of this paper is to summarize available options for rehabilitation after gynecological oncological diseases.

Optimal surgical treatment of locally advanced low rectal cancer.

Colorectal adenocarcinoma is the second leading cause of cancer deaths in Western countries. Rectal cancer comprises approximately 25% of the malignancies arising in the large bowel. However, the past two decades have seen many major advances in the diagnosis and treatment of this disease. While surgery is still the cornerstone of curative therapy, a multidisciplinary approach including neoadjuvant chemoradiotherapy has resulted in significantly improved outcomes. Information concerning the T, N, M stage and the exact location of tumor in relation to the anal verge are of crucial importance when planning a curative rectal cancer resection. Preoperative staging, utilizing a combination of diagnostic modalities, must be undertaken to determine whether or not neoadjuvant therapy is indicated. In radical resection of locally advanced low rectal cancer, several unique surgical management issues should be considered: 1) total mesorectal excision (TME); 2) longitudinal and circumferential resection margins; 3) autonomic nerve preservation (ANP); 4) sphincter preservation versus abdominoperineal resection (APR); 5) restoration of bowel continuity; and 6) laparoscopic versus open resection. The surgeon must first strive to achieve an oncologic cure, but whenever possible this should be undertaken with the goal of maintaining the patient's quality of life. The purpose of this review is to outline the critical surgical issues involved in management of locally advanced low rectal cancer.

Cell-specific expression of alpha 1(I) collagen-hGH minigenes in transgenic mice.

Sequences within the first intron of the alpha 1(I) collagen gene have been implicated in the regulation of expression of alpha 1(I) collagen-reporter gene constructs in cultured cells. However, the physiological significance of these intronic elements has not been established. We have used in situ hybridization to examine whether a cell-specific pattern of expression of human alpha 1(I) collagen-human growth hormone minigenes exists in transgenic mice. Our results indicate that transgenes which contained 2,300 bp of promoter/5' flanking sequence and an intact first intron were well expressed by fibroblasts in dermis and fascia, whereas transgenes lacking the intronic sequence, +292 to +1440, were not expressed in dermis and poorly expressed in fascia. Analysis of transgene expression in cultured fibroblasts obtained from dermal explants of transgenic animals confirmed the requirement for these intronic sequences in the regulation of the alpha 1(I) collagen gene. In contrast, transgenes with or without the intronic deletion were expressed equally well in tendon and bone, in a manner comparable to the endogenous mouse alpha 1(I) collagen gene, and expression of neither transgene was detected in skeletal muscle or perichondrium. These data support a model in which cis-acting elements in the first intron, and their cognate DNA-binding proteins, mediate transcription of the alpha 1(I) collagen gene in some cells, such as dermal fibroblasts, but not in tendon cells or osteoblasts. Moreover, regions of the gene not included in the sequence, -2300 to +1440, appear to be required for transcription in tissues such as skeletal muscle and perichondrium.

An upstream regulatory region mediates high-level, tissue-specific expression of the human alpha 1(I) collagen gene in transgenic mice.

Studies in vitro have not adequately resolved the role of intronic and upstream elements in regulating expression of the alpha 1(I) collagen gene. To address this issue, we generated 12 separate lines of transgenic mice with alpha 1(I) collagen-human growth hormone (hGH) constructs containing different amounts of 5'-flanking sequence, with or without most of the first intron. Transgenes driven by 2.3 kb of alpha 1(I) 5'-flanking sequence, whether or not they contained the first intron, were expressed at a high level and in a tissue-specific manner in seven out of seven independent lines of transgenic mice. In most tissues, the transgene was expressed at levels approaching that of the endogenous alpha 1(I) gene and was regulated identically with the endogenous gene as animals aged. However, in lung, expression of the transgene was anomalously high, and in muscle, expression was lower than that of the endogenous gene, suggesting that in these tissues other regions of the gene may participate in directing appropriate expression. Five lines of mice were generated containing transgenes driven by 0.44 kb of alpha 1(I) 5'-flanking sequence (with or without the first intron), and expression was detected in four out of five of these lines. The level of expression of the 0.44-kb constructs in the major collagen-producing tissues was 15- to 500-fold lower than that observed with the longer 2.3-kb promoter. While transgenes containing the 0.44-kb promoter and the first intron retained a modest degree of tissue-specific expression, those without the first intron lacked tissue specificity and were poorly expressed in all tissues except lung. These results contribute to our understanding of the role of the first intron in regulating alpha1(I) gene expression and identify a region, upstream of the basal alpha1(I) promotor, which is necessary for full tissue-specific, developmentally regulated expression of the alpha1(I) collagen gene.

Interactions between the promoter and first intron are involved in transcriptional control of alpha 1(I) collagen gene expression.

The first intron of the human collagen alpha 1(I) gene contains several positively and negatively acting elements. We have studied the transcription of collagen-human growth hormone fusion genes, containing deletions and rearrangements of collagen intronic sequences, by transient transfection of chick tendon fibroblasts and NIH 3T3 cells. In chick tendon fibroblasts, but not in 3T3 cells, inversion of intronic sequences containing a previously studied 274-base-pair segment, A274, resulted in markedly reduced human growth hormone mRNA levels as determined by an RNase protection assay. This inhibitory effect was largely alleviated when deletions were introduced in the collagen promoter of plasmids containing negatively oriented intronic sequences. Evidence for interaction of the promoter with the intronic segment, A274, was obtained by gel mobility shift assays. We suggest that promoter-intron interactions, mediated by DNA-binding proteins, regulate collagen gene transcription. Inversion of intronic segments containing critical interactive elements might then lead to an altered geometry and reduced activity of a transcriptional complex in those cells with sufficiently high levels of appropriate transcription factors. We further suggest that the deleted promoter segment plays a key role in directing DNA interactions involved in transcriptional control.

Human endothelial colony-forming cells expanded with an improved protocol are a useful endothelial cell source for scaffold-based tissue engineering.

One of the major challenges in tissue engineering is to supply larger three-dimensional (3D) bioengineered tissue transplants with sufficient amounts of nutrients and oxygen and to allow metabolite removal. Consequently, artificial vascularization strategies of such transplants are desired. One strategy focuses on endothelial cells capable of initiating new vessel formation, which are settled on scaffolds commonly used in tissue engineering. A bottleneck in this strategy is to obtain sufficient amounts of endothelial cells, as they can be harvested only in small quantities directly from human tissues. Thus, protocols are required to expand appropriate cells in sufficient amounts without interfering with their capability to settle on scaffold materials and to initiate vessel formation. Here, we analysed whether umbilical cord blood (CB)-derived endothelial colony-forming cells (ECFCs) fulfil these requirements. In a first set of experiments, we showed that marginally expanded ECFCs settle and survive on different scaffold biomaterials. Next, we improved ECFC culture conditions and developed a protocol for ECFC expansion compatible with 'Good Manufacturing Practice' (GMP) standards. We replaced animal sera with human platelet lysates and used a novel type of tissue-culture ware. ECFCs cultured under the new conditions revealed significantly lower apoptosis and increased proliferation rates. Simultaneously, their viability was increased. Since extensively expanded ECFCs could still settle on scaffold biomaterials and were able to form tubular structures in Matrigel assays, we conclude that these ex vivo-expanded ECFCs are a novel, very potent cell source for scaffold-based tissue engineering.

Analysis of the promoter and transcription start sites of the human thrombospondin 2 gene (THBS2).

To identify features of the human thrombospondin 2 gene (THBS2) important for regulation of expression, the sequences of 5 kb of the promoter/5' flank and 3 kb of transcribed and intronic DNA were determined. Two repetitive sequences were found: an MLT1c element located 2.2 kb 5' of exon 1 and, further 5', 1.8 kb of a Tigger1 element. Putative transcription factor binding sites that might be significant for THBS2 regulation included p53, NF-kappaB, Spl, Myc-CF1, NF-Y, CF1, AP1, and GATA sites. Alignment of the promoter/5' flank sequence with the mouse Thbs2 promoter revealed 78% identity for a 450 bp region immediately upstream from the mouse transcription start site. No significant homology was detected between the human thrombospondin 2 and thrombospondin 1 promoters. Comparison of the THBS2 genomic and cDNA sequences revealed that, in contrast to Thbs2, exon 1 is divided into exons 1A and 1B by a small (93 bp) intron. The transcription start site was investigated by a PCR procedure and by 5' RACE, and yielded a size for exon 1A of at least 186 bp. Tissue-specific differences in transcription start sites were found, with transcript lengths in the order: fetal lung > adult lung > fetal brain. These results suggest that tissue-specific differences in expression of the THBS2 gene may be determined, in part, by selection of the transcription start site and resulting differences in the 5' untranslated region.

Regulation of expression of the type I collagen genes.

The identification and functional analysis of DNA-protein interactions in the intronic and 5' flanking regions of the type I collagen genes has begun to define a series of cis-elements and trans-acting factors which regulate transcription of these genes. Studies such as these will eventually be expected to elucidate the mechanisms responsible for coordinate transcription of the alpha 1 and alpha 2 genes, a question which remains central to the field of collagen research. Although it is relatively straightforward to define sites of DNA-protein binding, interpretation of the functional importance of such interactions can be extremely complex. Furthermore, while mutation or deletion of a particular binding site may alter the functional activity of a construct transfected into cultured cells, there is no guarantee that a similar change will have the same effect in vivo, where the entire gene locus is present in its native chromosomal context. Nevertheless, these kinds of in vitro studies offer the best current approach to defining and isolating transcription factors that control expression of the alpha 1 and alpha 2 genes. Ultimately, it will be necessary to test the activity of such factors (and their respective cis-elements) in defined systems in vivo.