Search

Introdução: A semiologia médica é uma área de estudo acerca dos sinais e sintomas que um paciente pode apresentar, que aborda o desenvolvimento da relação médico-paciente mediante o exame clínico e a anamnese. Dessa forma, a monitoria em semiologia médica se constitui de suma importância para o desenvolvimento de novas habilidades e de raciocínio clínico do corpo discente. Objetivo: Relatar a experiência desenvolvida na atividade de monitoria em semiologia médica. Método: Trata-se de um estudo descritivo, do tipo relato de experiência, realizado a partir da vivência de monitoria de habilidades clínicas I (primeiro semestre) no segundo semestre de 2021 e no primeiro semestre de 2022. Discussão: A possibilidade do contato com a docência de habilidades clínicas permite aos monitores o aprimoramento dos conhecimentos em semiologia médica, acolhimento dos alunos de modo individualizado e a busca por novas experiências de qualificação acadêmica. Conclusão: A monitoria se mostra fundamental nesse processo de ensino-aprendizado da semiologia e contribui não só para o conhecimento dos discentes, como também para quem realiza a monitoria, constituindo um aprendizado de grande relevância durante o período acadêmico.

Background Breast magnetic resonance imaging (MRI) has high sensitivity in detecting invasive neoplasms. Controversy remains about its impact on the preoperative staging of breast cancer surgery. This study evaluated survival and surgical outcomes of preoperative MRI in conservative breast cancer surgery. Methods A phase III, randomized, open-label, single-center trial including female breast cancer participants, stage 0–III disease, and eligible for breast-conserving surgery. We compared the role of including MRI in preoperative evaluation versus radiologic exam routine with mammography and ultrasound in breast cancer conservative candidates. The primary outcome was local relapse-free survival (LRFS), and secondary outcomes were overall survival (OS), mastectomy rate, and reoperation rate. Results 524 were randomized to preoperative MRI group (n = 257) or control group (n = 267). The survival analysis showed a 5.9-years LRFS of 99.2% in MRI group versus 98.9% in control group (HR = 0.72; 95% CI 0.12—4.28; p = 0.7) and an OS of 95.3% in the MRI group versus 96.3% in the control group (HR = 1.37 95% CI 0.59–3.19; p = 0.8). Surgical management changed in 21 ipsilateral breasts in the MRI group; 21 (8.3%) had mastectomies versus one in the control group. No difference was found in reoperation rates, 22 (8.7%) in the MRI group versus 23 (8.7%) in the control group (RR = 1.002; 95% CI 0.57–1.75; p = 0.85). Conclusion Preoperative MRI increased the mastectomy rates by 8%. The use of preoperative MRI did not influence local relapse-free survival, overall survival, or reoperation rates.

International Dermatology Outcome Measures and the American Academy of Dermatology recently agreed on a physician-reported global severity measure to demonstrate quality of care in inflammatory dermatoses.Because patient-reported outcome measures are also important, we aimed to achieve consensus on 1 of these measures for clinical practice.Patients and providers participated in a consensus-building study involving a modified-Delphi technique. Voting was focused on identifying a minimal set of assessments for clinical practice, patient global assessments (PtGAs), Skindex instruments, and final instrument selection for quality improvement.Among 53 stakeholders,70% agreed that identification of patient goals, assessment of treatment harm, and assessment of the adequacy of treatment response were the minimal assessments needed for clinical practice. The most preferred PtGA was a 5-point PtGA (scale 0-4; 0 = clear, 4 = severe) having an optional check-box for worst ever. A new metric to assess change since treatment initiation, which we called a trajectory measure, was proposed. Stakeholders preferred Skindex instruments over PtGAs and a trajectory measure for clinical practice.A small number of patients and caregivers were involved and a consensus was not reached on all items.PtGAs as standalone measures do not adequately capture the patient's assessment of disease severity or the effect of care. The combination of a PtGA with a Skindex instrument or another measure of health-related quality of life might provide a more comprehensive evaluation of patients in clinical practice.

Women with premature ovarian insufficiency (POI) are exposed to a long period of estrogenic deficiency, which potentially brings higher health risks, especially regarding bone health. We performed a systematic review of the literature to evaluate the effect of hormone therapy (HT) on bone mineral density (BMD) in women with POI.A systematic search was performed of the MEDLINE and EMBASE databases up to September 2021. We included studies that analyzed women with idiopathic (spontaneous) POI treated with HT, and those who had BMD evaluated. Analysis of risk of bias of studies selected was performed.We found 335 articles and selected 16 studies according to the inclusion criteria. Most of the studies revealed lower bone density in both the femoral neck and lumbar spine of women with POI compared with healthy women. Bone mass had the tendency to remain stable in women treated with estrogen + progestin therapy. However, in women already with bone mass loss, the therapy - in the doses most frequently used - was not able to revert the loss. Higher doses of estrogen seem to have a positive impact on BMD, as did combined oral contraceptives used continuously. Also, the interruption of HT for longer than one year was linked to significant bone loss.Although HT brings clear benefits, further studies are needed to establish its long-term effects, as well as doses and formulations with better protective effects on the bone mass of these women.

The aim of our study was to evaluate the effects of the LNG-IUS on uterine volume, bleeding patterns, and LNG-IUS-related outcomes among women using the device to treat abnormal uterine bleeding caused by fibroids, adenomyosis, HMB (without structural cause), or contraception.This was a 5-year cohort study with LNG-IUS users. We selected 147 women, who were allocated to four groups: a) control (contraception indication); b) fibroids; c) adenomyosis; d) HMB. The visits for clinical and ultrasound evaluations were made at baseline and at 3, 6, 12, 24, 36, 48, and 60 months postinsertion. All data are expressed as mean and standard deviation (SD) or absolute and relative (%) frequency. Differences among groups were established by using the χ2 (chi-square) test and Fisher's exact tests for categorical outcomes, as well as the Mann-Whitney and the Kruskal-Wallis tests and Friedman's ANOVA for continuous variables. We used the 5% significance level as an indication of statistical significance. Logistic regression analyses were performed to study the association between predictors and outcomes. Results are expressed as odds ratios (ORs) with a 95% confidence interval (CI 95%).Although all groups had real rates of bleeding patterns, troublesome bleeding appeared to be more frequent in the fibroid group (∼15%). Also, along the 60 months of follow-up, uterine volume slightly decreased in the groups of HMB, adenomyosis, and fibroids, but not in the contraception group. However, the isolated volume of fibroids remained unchanged. In this cohort, we observed high continuation rates among LNG-IUS users. The uterine volume ≥200 cmThe LNG-IUS may control uterine menstrual bleeding as well as uterine volume in adenomyosis, fibroids, and HMB. An initial uterine volume smaller than 200 cm

Psoriasis studies increasingly employ outcomes that indicate complete disease resolution, yet remission and cure are poorly defined for psoriasis. We conducted a systematic literature review to identify definitions of psoriasis remission and cure reported in the literature. Medline, EMBASE, and The Cochrane Central Register of Controlled Trials databases were searched on July 22, 2020, for full-text studies providing definitions for psoriasis remission/cure. Definitions were analysed descriptively for endpoint, time-frame, on/off treatment, patient-reported outcomes, and disease domains. We identified 106 studies that provided 41 unique remission definitions. Most definitions included endpoints based on Psoriasis Area and Severity Index (PASI), such as PASI75 (n = 16 studies), PASI90 (n = 10), PASI100 (n = 10), and PASI of 0 (n = 3), and descriptive endpoints related to 'skin clearance' (n = 18). Few definitions specified time-frame, on/off treatment or other psoriasis-related disease domains. One small consensus-initiative defined drug-free remission for plaque psoriasis by BSA of 0 without any therapy for at least 12 months. While there is no cure for psoriasis, seven studies defined psoriasis cure using similar endpoints to those used to define remission. We identified a variety of definitions of psoriasis remission. These results will inform the development of consensus-based definitions for psoriasis remission to support efforts to improve research and clinical outcomes.

Background The presence of Extracapsular Extension (ECE) in the Sentinel Lymph Node Biopsy (SLNB) is still a doubt in the literature. Some studies suggest that the presence of ECE may be related to a greater number of positive axillary lymph nodes which could impact Disease Free Survival (DFS) and Overall Survival (OS). This study searches for the clinical significance of the ECE. Methods Retrospective cohort comparing the presence or absence of ECE in T1-2 invasive breast cancer with positive SLNB. All cases treated surgically at the Cancer Institute of the State of São Paulo (ICESP) between 2009 and 2013 were analyzed. All patients with axillary disease in SLNB underwent AD. Outcomes Identify the association between the presence and length of ECE and additional axillary positive lymph nodes, OS and DFS between both groups. Results 128 patients with positive SLNB were included, and 65 had ECE. The mean metastasis size of 0.62 (SD = 0.59) mm at SLNB was related to the presence of ECE (p < 0.008). The presence of ECE was related to a higher mean of positive sentinel lymph nodes, 3.9 (± 4.8) vs. 2.0 (± 2.1), p = 0.001. The median length of follow-up was 115 months. The OS and DFS rates had no differences between the groups. Conclusion The presence of ECE was associated with additional positive axillary lymph nodes in this study. Therefore, the OS and DFS were similar in both groups after 10 years of follow-up. It is necessary for additional studies to define the importance of AD when SLNB with ECE.

Background Venous Thromboembolism (VTE) is an important cause of morbidity in cancer patients. Breast cancer patients undergoing surgical treatment are at an increased risk of VTE. The aim of this study was to determine the frequency of VTE in patients who underwent surgery for the treatment of breast cancer and to identify the related risk factors. Methods A historical cohort of patients at the São Paulo State Cancer Institute (ICESP) underwent surgery for breast cancer. The inclusion criteria covered patients with invasive breast cancer or ductal carcinoma in situ who had breast surgery anytime from January 2016 to December 2018. Results Of the 1672 patients included in the study, 15 had a confirmed diagnosis of VTE (0.9%), and 3 of these had deep vein thrombosis (0.2%), and 12, had pulmonary thromboembolism (0.7%). Clinical and tumoral characteristics did not differ between the groups. The incidence of VTE was higher in patients who had undergone skin-sparing mastectomy or nipple-sparing mastectomy (p = 0.032). Immediate reconstruction, particularly with abdominal-based flaps (4.7%), increased VTE events (p = 0.033). Median surgical time was higher in patients with VTE episodes (p = 0.027), and total hospital length of stay increased in days (6 days vs. 2 days, p = 0.001). Neoadjuvant chemotherapy and postoperative prophylaxis with Low Molecular Weight Heparin (LMWH) were associated with lower VTE rates (0.2% vs. 1.2%, p = 0.048 and 0.7% vs. 2.7%, p = 0.039; respectively) in these patients. Conclusions The incidence of VTE events in breast cancer patients who underwent surgery was 0.9%. Immediate reconstruction (especially with abdominal-based flaps), skin-sparing/nipple-sparing mastectomies, and longer surgeries were associated with increased risk. The LMWH postoperative prophylaxis reduced this risk.

Unprecedented domino aza-annulations of (

Channel equalization is the efficient method for recovering distorted signal and correspondingly reducing bit error rate (BER). Different type of equalizations, like feed forward equalization (FFE) and decision feedback equalization (DFE) are canceling channel effect and recovering channel response. Separate optimization of tap coefficients for FFE and DFE does not give optimal result. In this case FFE and DFE tap coefficients are found separately and they are not collaborating. Therefore, the final equalization result is not global optimal. In the present paper new analytical method for finding best tap coefficients for FFE and DFE joint equalization is introduced. The proposed method can be used for both NRZ and PAM4 signals. The idea of the methodology is to combine FFE and DFE tap coefficients into one optimization problem and allow them to collaborate and lead to the global optimal solution. The proposed joint optimization method is fast, easy to implement and efficient. The method has been tested for several measured channels and the analysis of the results are discussed.

International Dermatology Outcome Measures (IDEOM) is a non-profit organization founded in 2013. It is composed of researchers and stakeholders who work to develop evidenced-based outcome measures to enhance research and treatment recommendations of dermatologic diseases.The 2021 IDEOM Virtual Annual Meeting occurred from November 19-20, 2021. Contributions were made by leaders and stakeholders from the psoriasis, psoriatic arthritis, pediatric hidradenitis suppurativa, acne, vitiligo, actinic keratosis, alopecia areata, itch, and cutaneous lymphoma workgroups. The psoriasis, psoriatic arthritis, and actinic keratosis workgroups provided an overview of their respective instruments for treatment satisfaction and symptom measurement. The inaugural meetings of the itch, alopecia areata, and cutaneous lymphoma workgroups identified unmet needs of their respective diseases and future goals. The acne, vitiligo, and pediatric hidradenitis suppurativa workgroups discussed concerns of quality of life, instruments for symptom measurement, and screening tools. Additionally, a representative from the US Food and Drug Administration was in attendance and presented an update on topical drugs and generics. This report provides a summary of workgroup updates from the past year and future directions established during the meeting.This report summarizes progress made by each IDEOM workgroup at the 2021 IDEOM Virtual Annual Meeting. J Drugs Dermatol. 2022;21(8):867-874. doi:10.36849/JDD.6974.

The hydrofoil is one of the typical factors that affect the vortex structure and flow characteristics of hydraulic machinery. In order to enhance the utilization efficiency of hydraulic machinery in marine energy, parallel grooves are proposed and applied to the hydrofoil. Following that, a numerical analysis is performed using the SST k- turbulence model, and the effects of hydrofoil profile, angle of attack, and flow are investigated. The profiles of NACA 0066, NACA 8412, NACA M2, and RAE 104 are considered for the study. The performance is analyzed based on the lift to drag ratio. The best model from this is given with surface modification and the flow study is carried out at different angles of attack. The modified profile of NACA 8412 with parallel groves has shown the highest lift to drag ratio at a 12-degree angle of attack.

Este artigo busca tecer reflexões a partir de práticas de resistência religiosa e política que constroem uma relação integrativo-subversiva e não dicotômica entre dissidências sexuais e de gênero e religiosidade cristã. Para isso, serão lançadas discussões que passam desde a teoria da secularização enquanto movimento ideológico, até o projeto colonial que sufoca e impossibilita a diversidade de manifestações religiosas que articulam-se com o exercício político de resistência sexo-gênero dissidente de existir. Tendo como base teórico-política o pensamento decolonial, pretende-se defender como a construção de uma posição cristã dissidente tensiona tanto os cânones religiosos, quanto o ideário moderno/colonial de progresso, anunciando saberes e práticas fronteiriças que (re)constroem a experiência religiosa e a reivindicam para além de um grande monólito.

The high prevalence of pelvic organ prolapse (POP) in women requires attention and constant review of treatment options. Sacrospinous ligament fixation (SSLF) for apical prolapse has benefits, high efficacy, and low cost. Our objective is to compare anterior and posterior vaginal approach in SSLF in relation to anatomical structures and to correlate them with body mass index (BMI).Sacrospinous ligament fixation was performed in fresh female cadavers via anterior and posterior vaginal approaches, using the CAPIO®SLIM device (Boston Scientific, Natick, MA, USA). The distances from the point of fixation to the pudendal artery, pudendal nerve, and inferior gluteal artery were measured.We evaluated 11 cadavers with a mean age of 70.1 ± 9.9 years and mean BMI 22.4 ± 4.6 kg/mThere was no difference in the measurements obtained in the anterior and posterior vaginal approaches. A direct correlation between BMI and the distances to the pudendal artery and pudendal nerve was found.

Tuberous Sclerosis Complex (TSC) tumors are presently incurable despite a cytostatic response to mTOR pathway inhibition since recurrence of disease occurs after discontinuation of treatment. Here, we explored the hypothesis that inhibiting tyrosine kinase activity in mesenchymal lineage specific platelet derived growth factor receptor beta (PDGFRβ) signaling in TSC tumors is cytocidal and attenuates tumorigenesis at significantly higher levels than treatment with an mTOR inhibitor. Rapamycin- versus tyrosine kinase inhibitor (TKI)-induced renal angiomyolipoma (AML) and pulmonary lymphangioleiomyomatosis (LAM) tumor cells were comparatively analyzed using cell survival assays, and RNA sequencing and bioinformatics to distinguish tumoricidal mechanisms adopted by each drug type. The efficacy of imatinib therapy was validated against spontaneously developing renal cystadenomas in Tuberous Sclerosis Tsc2+/- mouse models (C57BL/6J mice; N=6; 400mg/kg/day; oral gavage) compared to Tsc2+/- mice treated with phosphate buffered saline (PBS) (C57BL/6J mice; N=6). Our study revealed that TKIs imatinib and nilotinib were cytocidal to both pulmonary LAM and renal AML cell cultures through the downregulation of the glycoprotein GPVI pathway and resultant disruption in mitochondrial permeability, increased cytosolic cytochrome C, and caspase 3 activation. Importantly, renal tumor growth was significantly attenuated in imatinib-treated Tsc2+/- mice compared to PBS treatment. The preclinical studies reported here provide evidence documenting the effectiveness of TKIs in limiting LAM and AML cell growth and viability with important clinical potential. Furthermore, these drugs elicit their effects by targeting a PDGF pathway-dependent apoptotic mechanism supporting the investigation of these drugs as a novel class of TSC therapeutics.

We propose a self-supervised shared encoder model that achieves strong results on several visual, language and multimodal benchmarks while being data, memory and run-time efficient. We make three key contributions. First, in contrast to most existing works, we use a single transformer with all the encoder layers processing both the text and the image modalities. Second, we propose a stage-wise training strategy where the model is first trained on images, then jointly with unimodal text and image datasets and finally jointly with text and text-image datasets. Third, to preserve information across both the modalities, we propose a training pipeline that learns simultaneously from gradient updates of different modalities at each training update step. The results on downstream text-only, image-only and multimodal tasks show that our model is competitive with several strong models while using fewer parameters and lesser pre-training data. For example, MoMo performs competitively with FLAVA on multimodal (+3.1), image-only (+1.1) and text-only (-0.1) tasks despite having 2/5th the number of parameters and using 1/3rd the image-text training pairs. Finally, we ablate various design choices and further show that increasing model size produces significant performance gains indicating potential for substantial improvements with larger models using our approach.

Plants are sessile, and their ability to reprogram their metabolism to adapt to fluctuations in soil water level is crucial but not clearly understood. A study was performed to determine alterations in intermediate metabolites involved in central carbon metabolism (CCM) following exposure of Mexican mint (Plectranthus amboinicus) to varying watering regimes. The water treatments were regular watering (RW), drought (DR), flooding (FL), and resumption of regular watering after flooding (DHFL) or after drought (RH). Leaf cluster formation and leaf greening were swift following the resumption of regular watering. A total of 68 key metabolites from the CCM routes were found to be significantly (p < 0.01) impacted by water stress. Calvin cycle metabolites in FL plants, glycolytic metabolites in DR plants, total tricarboxylic acid (TCA) cycle metabolites in DR and DHFL plants, and nucleotide biosynthetic molecules in FL and RH plants were significantly (p < 0.05) increased. Pentose phosphate pathway (PPP) metabolites were equally high in all the plants except DR plants. Total Calvin cycle metabolites had a significantly (p < 0.001) strong positive association with TCA cycle (r = 0.81) and PPP (r = 0.75) metabolites. Total PPP metabolites had a moderately positive association with total TCA cycle metabolites (r = 0.68; p < 0.01) and a negative correlation with total glycolytic metabolites (r = −0.70; p < 0.005). In conclusion, the metabolic alterations of Mexican mint plants under different watering regimes were revealed. Future studies will use transcriptomic and proteomic approaches to identify genes and proteins that regulate the CCM route.

Methionine deprivation stress (MDS) eliminates mitotic activity in melanoma cells regardless of stage, grade, or TP53 status, whereas it has a negligible effect on normal skin fibroblasts. In most cases, apoptosis accounts for the elimination of up to 90% of tumor cells from the culture within 72 hours after MDS, leaving a scattered population of multinucleated resistant cells. Loss of mitosis in tumor cells is associated with marked reduction of cyclin-dependent kinase (CDK) 1 transcription and/or loss of its active form (CDK1-P-Thr161), which is coincident with up-regulation of CDKN1A, CDKN1B, and CDKN1C (p21, p27, and p57). Expression of the proapoptotic LITAF, IFNGR, EREG, TNFSF/TNFRSF10 and TNFRSF12, FAS, and RNASEL is primarily up-regulated/induced in cells destined to undergo apoptosis. Loss of Aurora kinase B and BIRC5, which are required for histone H3 phosphorylation, is associated with the accumulation of surviving multinucleated cells. Nevertheless, noncycling survivors of MDS are sensitized to temozolomide, carmustin, and cisplatin to a much greater extent than normal skin fibroblasts possibly because of the suppression of MGMT/TOP1/POLB, MGMT/RAD52/RAD54, and cMET/RADD52, respectively. Sensitivity to these and additional genotoxic agents and radiation may also be acquired due to loss of cMET/OGG1, reduced glutathione reductase levels, and a G2-phase block that is a crucial step in the damage response associated with enhancement of drug toxicity. Although the genes controlling mitotic arrest and/or apoptosis in response to low extracellular methionine levels are unknown, it is likely that such control is exerted via the induction/up-regulation of tumor suppressors/growth inhibitor genes, such as TGFB, PTEN, GAS1, EGR3, BTG3, MDA7, and the proteoglycans (LUM, BGN, and DCN), as well as the down-regulation/loss of function of prosurvival genes, such as NFκB, MYC, and ERBB2. Although MDS targets several common genes in tumors, mutational variability among melanomas may decide which metabolic and signal transduction pathways will be activated or shutdown. (Mol Cancer Res 2006;4(8):575–89)

Supplementary Table S1 from Mitotic Arrest, Apoptosis, and Sensitization to Chemotherapy of Melanomas by Methionine Deprivation Stress

Supplementary Fig. S2 from Adenoviral endoplasmic reticulum–targeted mda-7/interleukin-24 vector enhances human cancer cell killing

We developed several adenoviral vectors designed to target MDA-7 expression to different subcellular compartments [endoplasmic reticulum (ER), mitochondria, nucleus, and cytosol] and evaluated their ability to enhance apoptosis. Adenoviral ER-targeted mda-7/interleukin-24 vector (Ad-ER-mda7) selectively and effectively inhibited the growth and proliferation of lung (A549 and H1299) and esophageal (Seg1 and Bic1) cancer cells by enhancing cell killing. Both Ad-mda7 and Ad-ER-mda7 activated a novel pathway of ER stress-induced apoptosis characterized by unregulated expression of phosphorylated JNK, phosphorylated c-Jun, and phosphorylated RNA-dependent protein kinase. Caspase-4 activation mediated Ad-mda7- and Ad-ER-mda7-induced cell death. In addition, Ad-mda7- and Ad-ER-mda7-mediated growth inhibition correlated with activation of ER molecular markers RNA-dependent protein kinase and JNK both in vitro (in Ad-mda7- or Ad-ER-mda7-treated lung cancer cells) and in vivo. These findings suggest that vectors targeting the ER (Ad-ER-mda7) may be more effective in cancer gene therapy possibly through more effective induction or ER stress pathways. [Mol Cancer Ther 2008;7(8):2528–35]

Supplementary Fig. S1 from Adenoviral endoplasmic reticulum–targeted mda-7/interleukin-24 vector enhances human cancer cell killing

Supplementary Figure 10 - PDF file 84K, HMGA2 mRNA expression in MCF7 and MDA-MB231 human breast cancer cells treated with TGFBeta 1 as detected by qRT-PCR

Legend for Supplemental Figures S1-S2.

Five Supplementary Figures from Modulation of Gene Expression in Human Central Nervous System Tumors under Methionine Deprivation-induced Stress

Supplementary Figure 6 - PDF file 132K, (A) HMGA2 was detected at the edge of the metastatic lung tumor (left, arrows) and from the vessel of the liver stroma to the parenchyma in the metastatic liver tumor (right, arrows). Scale bars represent 100 microm. (B) Liver metastases (arrows) were induced in 4TO7 cells stably over-expressing HMGA2 (4TO7-HMGA2)

Tuberin is present in all Tsc2+/- mesenchymal tumors.

Tsc2+/- extra-renal tumorigenesis is HMGA2 dependent (S1); Summary of human AML LOH analysis (S2).

Supplementary Figure 3 - PDF file 171K, Immunofluorescence analysis of ZEB1 (left), Fibronectin (right) expression in MCF7-Mock and MCF7-HMGA2 cells. Scale bars represent 50 microm. ZEB1 and fibronectin are expressed in the nucleus of MCF7-HMGA2 cells that have undergone the EMT as is consistent with the other EMT markers in Figure 1C

Supplementary Methods from Activation of the Fas-FasL Signaling Pathway by MDA-7/IL-24 Kills Human Ovarian Cancer Cells

Supplementary Figure 2 - PDF file 77K, Agarose Colony Formation Assay. Anchorage-independent proliferation was performed by plating SW403, HT29, SW620, SW480 and HCT116 (1000 cells per well) in six-well plates, with a bottom layer of 0.6% agar and a top layer of 0.3% agar containing the cells. After 21 days incubation, colonies were counted