Head and neck radiotherapy quality assurance conference for dedicated review of delineated targets and organs at risk: results of a prospective study.

Purpose: Head and neck (HN) radiotherapy (RT) is complex, involving multiple target and organ at risk (OAR) structures delineated by the radiation oncologist. Site-agnostic peer review after RT plan completion is often inadequate for thorough review of these structures. In-depth review of RT contours is critical to maintain high-quality RT and optimal patient outcomes. Materials and Methods: In August 2020, the HN RT Quality Assurance Conference, a weekly teleconference that included at least one radiation oncology HN specialist, was activated at our institution. Targets and OARs were reviewed in detail prior to RT plan creation. A parallel implementation study recorded patient factors and outcomes of these reviews. A major change was any modification to the high-dose planning target volume (PTV) or the prescription dose/fractionation; a minor change was modification to the intermediate-dose PTV, low-dose PTV, or any OAR. We analysed the results of consecutive RT contour review in the first 20 months since its initiation. Results: A total of 208 patients treated by 8 providers were reviewed: 86·5% from the primary tertiary care hospital and 13·5% from regional practices. A major change was recommended in 14·4% and implemented in 25 of 30 cases (83·3%). A minor change was recommended in 17·3% and implemented in 32 of 36 cases (88·9%). A survey of participants found that all (n = 11) strongly agreed or agreed that the conference was useful. Conclusion: Dedicated review of RT targets/OARs with a HN subspecialist is associated with substantial rates of suggested and implemented modifications to the contours.

Detection of CRISPR-mediated genome modifications through altered methylation patterns of CpG islands.

BACKGROUND: The development and application of CRISPR technologies for the modification of the genome are rapidly expanding. Advances in the field describe new CRISPR components that are strategically engineered to improve the precision and reliability of CRISPR editing within the genome sequence. Genome modification using induced genome breaks that are targeted and mediated by CRISPR components leverage cellular mechanisms for repair like homology directed repair (HDR) to incorporate genomic edits with increased precision. RESULTS: In this report, we describe the gain of methylation at typically hypomethylated CpG island (CGI) locations affected by the CRISPR-mediated incorporation of donor DNA using HDR mechanisms. With characterization of CpG methylation patterns using whole genome bisulfite sequencing, these CGI methylation disruptions trace the insertion of the donor DNA during the genomic edit. These insertions mediated by homology-directed recombination disrupt the generational methylation pattern stability of the edited CGI within the cells and their cellular lineage within the animal strain, persisting across generations. Our approach describes a statistically based workflow for indicating locations of modified CGIs and provides a mechanism for evaluating the directed modification of the methylome of the affected CGI at the CpG-level. CONCLUSIONS: With advances in genome modification technology comes the need to detect the level and persistence of methylation change that modifications to the genomic sequence impose upon the collaterally edited methylome. Any modification of the methylome of somatic or germline cells could have implications for gene regulation mechanisms governed by the methylation patterns of CGI regions in the application of therapeutic edits of more sensitively regulated genomic regions. The method described here locates the directed modification of the mouse epigenome that persists over generations. While this observance would require supporting molecular observations such as direct sequence changes or gene expression changes, the observation of epigenetic modification provides an indicator that intentionally directed genomic edits can lead to collateral, unintentional epigenomic changes post modification with generational persistence.

Contraception during Coronavirus-Covid 19 pandemia. Recommendations of the Board of the Italian Society of Contraception.

Purpose: The Italian Society of Contraception identified as one of its priorities the need to give recommendations on management of contraception during Coronavirus-Covid 19 pandemiaMaterials and methods: A concise communication was produced which summarises in an easy-to-read format suitable for clinicians the management of the different contraceptives mostly used. Information how to manage contraception in different conditions is presented.Results: Women may, in general, continue to use either intrauterine and or hormonal contraceptives. The use of condom should be added to any hormonal contraceptive, when the contraceptive efficacy is reduced or when women stop the contraceptive method.Conclusion: At the present time, during the Coronavirus-Covid 19 pandemia, no data contraindicate the use of intrauterine or hormonal contraceptives. Conversely the use of an appropriate contraception is advocate to prevent unintended pregnancies.

TIA: algorithms for development of identity-linked SNP islands for analysis by massively parallel DNA sequencing.

BACKGROUND: Single nucleotide polymorphisms (SNPs) located within the human genome have been shown to have utility as markers of identity in the differentiation of DNA from individual contributors. Massively parallel DNA sequencing (MPS) technologies and human genome SNP databases allow for the design of suites of identity-linked target regions, amenable to sequencing in a multiplexed and massively parallel manner. Therefore, tools are needed for leveraging the genotypic information found within SNP databases for the discovery of genomic targets that can be evaluated on MPS platforms. RESULTS: The SNP island target identification algorithm (TIA) was developed as a user-tunable system to leverage SNP information within databases. Using data within the 1000 Genomes Project SNP database, human genome regions were identified that contain globally ubiquitous identity-linked SNPs and that were responsive to targeted resequencing on MPS platforms. Algorithmic filters were used to exclude target regions that did not conform to user-tunable SNP island target characteristics. To validate the accuracy of TIA for discovering these identity-linked SNP islands within the human genome, SNP island target regions were amplified from 70 contributor genomic DNA samples using the polymerase chain reaction. Multiplexed amplicons were sequenced using the Illumina MiSeq platform, and the resulting sequences were analyzed for SNP variations. 166 putative identity-linked SNPs were targeted in the identified genomic regions. Of the 309 SNPs that provided discerning power across individual SNP profiles, 74 previously undefined SNPs were identified during evaluation of targets from individual genomes. Overall, DNA samples of 70 individuals were uniquely identified using a subset of the suite of identity-linked SNP islands. CONCLUSIONS: TIA offers a tunable genome search tool for the discovery of targeted genomic regions that are scalable in the population frequency and numbers of SNPs contained within the SNP island regions. It also allows the definition of sequence length and sequence variability of the target region as well as the less variable flanking regions for tailoring to MPS platforms. As shown in this study, TIA can be used to discover identity-linked SNP islands within the human genome, useful for differentiating individuals by targeted resequencing on MPS technologies.

Efficacy and adverse events of oral isotretinoin for acne: a systematic review.

Despite many years of clinical use of isotretinoin, a comprehensive review of evidence for isotretinoin therapy in patients with acne is lacking. We searched MEDLINE, Embase, Cochrane Central, relevant web pages and bibliographies for randomized controlled trials in acne evaluating isotretinoin vs. control (placebo or other therapy). Data were extracted and summarized descriptively. Eleven trials were identified (total 760 patients randomized), containing mostly men. Mean treatment ages ranged from 18 to 47·9 years and participants generally had moderate-to-severe acne. Across all trials, isotretinoin therapy reduced acne lesion counts by a clinically relevant amount, and always by a greater amount than control, which was either placebo (two studies), oral antibiotics (seven studies) or other control (two studies). Across trials with an overall low risk of bias, two of three demonstrated statistically significant differences between isotretinoin and control. The frequency of adverse events was twice as high with isotretinoin (751 events) than with control (388 events). More than half of all adverse events were dermatological and related to dryness. Adverse events from isotretinoin causing participant withdrawal from trials (12 patients) included Stevens-Johnson syndrome, cheilitis, xerosis, acne flare, photophobia, elevated liver enzymes, decreased appetite, headaches and depressed mood. This review suggests that isotretinoin is effective in reducing acne lesion counts, but adverse events are common. This study was registered with PROSPERO number CRD42015025080.

Mitrecin A, an endolysin-like bacteriolytic enzyme from a newly isolated soil streptomycete.

UNLABELLED: An open reading frame with homology to known endolysin genes was identified in the genome of Streptomyces sp. strain 212, which is a newly isolated soil bacterium. The heterologously expressed gene product of this endolysin-like gene, called Mitrecin A, demonstrated bacteriolytic activity against several Gram-negative bacteria. The genome of the bacterial strain was sequenced to draft quality using pyrosequencing followed by genome assembly and gene annotation. Within the sequence, a chromosomally located endolysin-like open reading frame was predicted. The gene product, designated Mitrecin A, was heterologously expressed and isolated from contaminating proteins as a fusion protein to a 6-histidine tag. Mitrecin A consists of 127 amino acids arranged in modular domains of activity. It has an estimated molecular weight of 14.3 kDa and retains sequence homology to the M15C peptidase subfamily of zinc metallocarboxypeptidases. The heat-labile purified recombinant protein has an overall positive charge, has optimal catalytic activities at 26°C in solution of pH 9 with 1% saline and has bacteriolytic activity against Gram-negative bacteria of the medically important genera Aeromonas, Escherichia, Salmonella, Shigella, Vibrio and Yersinia. SIGNIFICANCE AND IMPACT OF THE STUDY: The gene of a new protein antimicrobial, Mitrecin A, was discovered in the genome of a soil bacterium. The purified recombinant enzyme, resulting from heterologous over expression of the gene, was found to be tolerant of increased pH conditions and to have bacteriolytic activity against Gram-negative bacteria of the medically important genera Aeromonas, Escherichia, Salmonella, Shigella, Vibrio and Yersinia. Characterization of enzymes such as Mitrecin A from previously uncharacterized bacteria provides potential options for new biocontrol agents in medically and economically important applications like therapeutics, disinfectants, food preservatives, agricultural livestock antimicrobials, and inhibitors of biofilm production.

Unmet needs and knowledge of postpartum contraception in Italian women.

PURPOSE OF INVESTIGATION: Clinic visits during pregnancy and puerperium provide a unique opportunity to counsel women on contraception practices. With the aim of evaluating postpartum contraceptive attitudes among urban women attending an antenatal care center and delivering in the same facility, a structured questionnaire was administered to assess desired and received information on contraception in the postpartum period. RESULTS: A total of 436 consecutive interviews were collected during the study period. Pregnancy was unplanned in 39% of the women interviewed. Overall, 269 women (61.7%) had decided to use a method of family planning during postpartum. Among the 112 women who stated they did not want to use a method during postpartum, almost 50% stated that they "did not think they needed it", due to a perceived lack of real risk. Of the 436 women interviewed, only 5.5 % women acknowledged that they had received information on contraceptive use. CONCLUSION: The present study indicates a need for ante- and postpartum counseling of women even in urban areas of Italy.

Decision Regret among Patients with Early-stage Lung Cancer Undergoing Radiation Therapy or Surgical Resection.

AIMS: Clinical equipoise exists regarding early-stage lung cancer treatment among patients as trials comparing stereotactic body radiation therapy (SBRT) and surgical resection are unavailable. Given the potential differences in treatment effectiveness and side-effects, we sought to determine the associations between treatment type, decision regret and depression. MATERIALS AND METHODS: A multicentre, prospective study of patients with stage IA-IIA non-small cell lung cancer (NSCLC) with planned treatment with SBRT or surgical resection was conducted. Decision regret and depression were measured using the Decision Regret Scale (DRS) and Patient Health Questionnaire-4 (PHQ-4) at 3, 6 and 12 months post-treatment, respectively. Mixed linear regression modelling examined associations between treatment and decision regret adjusting for patient sociodemographics. RESULTS: Among 211 study participants with early-stage lung cancer, 128 (61%) patients received SBRT and 83 (39%) received surgical resection. The mean age was 73 years (standard deviation = 8); 57% were female; 79% were White non-Hispanic. In the entire cohort at 3 months post-treatment, 72 (34%) and 57 (27%) patients had mild and severe decision regret, respectively. Among patients who received SBRT or surgery, 71% and 46% of patients experienced at least mild decision regret at 3 months, respectively. DRS scores increased at 6 months and decreased slightly at 12 months of follow-up in both groups. Higher DRS scores were associated with SBRT treatment (adjusted mean difference = 4.18, 95% confidence interval 0.82 to 7.54) and depression (adjusted mean difference = 3.49, 95% confidence interval 0.52 to 6.47). Neither patient satisfaction with their provider nor decision-making role concordance was associated with DRS scores. CONCLUSIONS: Most early-stage lung cancer patients experienced at least mild decision regret, which was associated with SBRT treatment and depression symptoms. Findings suggest patients with early-stage lung cancer may not be receiving optimal treatment decision-making support. Therefore, opportunities for improved patient-clinician communication probably exist.

Trends in osteoporosis care patterns during the COVID-19 pandemic in Alberta, Canada.

PURPOSE/INTRODUCTION: The objective of this study was to describe osteoporosis-related care patterns during the coronavirus disease 2019 (COVID-19) pandemic in Alberta, Canada, relative to the 3-year preceding. METHODS: A repeated cross-sectional study design encompassing 3-month periods of continuous administrative health data between March 15, 2017, and September 14, 2020, described osteoporosis-related healthcare resource utilization (HCRU) and treatment patterns. Outcomes included patients with osteoporosis-related healthcare encounters, physician visits, diagnostic and laboratory test volumes, and treatment initiations and disruptions. The percent change between outcomes was calculated, averaged across the control periods (2017-2019), relative to the COVID-19 periods (2020). RESULTS: Relative to the average control March to June period, all HCRU declined during the corresponding COVID-19 period. There was a reduction of 14% in patients with osteoporosis healthcare encounters, 13% in general practitioner visits, 9% in specialist practitioner visits, 47% in bone mineral density tests, and 13% in vitamin D tests. Treatment initiations declined 43%, 26%, and 35% for oral bisphosphonates, intravenous bisphosphonates, and denosumab, respectively. Slight increases were observed in the proportion of patients with treatment disruptions. In the subsequent June to September period, HCRU either returned to or surpassed pre-pandemic levels, when including telehealth visits accounting for 33-45% of healthcare encounters during the COVID periods. Oral bisphosphonate treatment initiations remained lower than pre-pandemic levels. CONCLUSIONS: This study demonstrates the COVID-19 pandemic and corresponding public health lockdowns further heightened the "crisis" around the known gap in osteoporosis care and altered the provision of care (e.g., use of telehealth and initiation of treatment). Osteoporosis has a known substantial care and management disparity, which has been classified as a crisis. The COVID-19 pandemic created additional burden on osteoporosis patient care with healthcare encounters, physician visits, diagnostic and laboratory tests, and treatment initiations all declining during the initial pandemic period, relative to previous years.

Streptomyces scopuliridis sp. nov., a bacteriocin-producing soil streptomycete.

Actinomycete strain RB72(T) was isolated from woodland bluff soil in northern Alabama, USA, and shown to produce a broad spectrum bacteriocin. Based on morphological and chemotaxonomic characteristics, the strain was determined to belong to the genus Streptomyces. Phylogenetic analysis of the near-complete 16S rRNA gene sequence indicated that it differed from those of the described streptomycetes available in public databases. The distinctive white aerial hyphae and lack of sporulation suggest a deficiency in the whi pathway of the organism. A combination of substrate utilization patterns, morphological and chemotaxonomic characteristics and DNA-DNA hybridization results supported the affiliation of strain RB72(T) to the genus Streptomyces and enabled the genotypic and phenotypic differentiation of strain RB72(T) from closely related reference strains. Strain RB72(T) therefore represents a novel species of the genus Streptomyces, for which the name Streptomyces scopuliridis sp. nov. is proposed. The type strain is RB72(T) ( = DSM 41917(T)  = NRRL B-24574(T)).

Mirena, an Italian experience.

AIM: The aim of this review was to evaluate contraceptive efficacy, compliance, effect on menstrual cycle of a levonorgestrel-releasing device (Mirena®) in an Italian setting. METHODS: During the 48 months of recruitment, 128 women accepted the insertion of the LNG-IUS satisfying all inclusion criteria; 71.6% of the women completing five years of use had heavy menstrual blood loss. General and pelvic examination, including an ultrasound scan were performed at recruitment and repeated at 1, 3, 6, months postinsertion. Each subject was given a menstrual diary to report both menstrual patterns and side effects. RESULTS: Over the 7 308 woman/months of observation, no pregnancy occurred. Menstrual blood flow decreased in all patients, both in terms of quantity and duration. In the vast majority of cases (93.7%), spotting disappeared within six months; within the first six cycles postinsertion, amenorrhoea occurred in 29.5% of all women. In five subjects the device was removed before the planned five years of observation: two women desired a pregnancy and three, in spite of a pre-existing condition of menorrhagia, requested removal within the first nine months, because they could not tolerate amenorrhoea. Three expulsions and one dislocation of the device were also detected, all within the first eight months. CONCLUSION: Although in Italy intrauterine contraception is poorly accepted, once started on Mirena® women found that the device represents a safe and effective contraceptive modality, particularly indicated in the presence of heavy or prolonged bleeding. The only important side effect reported was breast tenderness; in addition the transient appearance of ovarian cystic images was observed.

[Voluntary pregnancy termination over the first trimester. A five-year observation]. / Interruzione volontaria di gravidanza oltre il primo trimestre. Cinque anni di osservazione.

AIM: The law for legalising abortion was approved by the Italian Government in May 1978. In regulating legal abortion this law identifies two different scenarios: one where legal abortion is performed within 90 days of gestational age, and the second where it can be performed beyond this term but within 120 days: ''when pregnancy or delivery can cause a severe damage to the woman's life, in case of severe pathologies, as fetal relevant anomalies or malformations which can cause a severe damage to the woman's physical or psychological health''. Since during the last years an increase of requests for voluntary pregnancy termination (VPT) over 90 days of gestational age has been observed in Italy, it was decided to carry out a retrospective study on the reasons for requesting such an operation. METHODS: All interventions for VPT over 90 days of gestational age performed in the Department of Obstetrics and Gynecology in the University of Rome ''La Sapienza'' between January 2003 and December 2007 have been re-assessed, analysing age of women, obstetric anamnesis, reasons for VPT request, gestational age, mode of intervention, complications due to intervention and days of inpatient admission. RESULTS: During five years 255 women demanded to terminate a pregnancy over the first trimester. In all cases requested have been authorized following a psychological consult assessing a severe damage on psychological health by the Clinical Psychology Service of ''La Sapienza'' University, that in all cases was subsequent to a diagnosis of fetal anomalies, ascertained by a genetic test and/or ultrasound scan. Anomalies were genetic in 112 of cases (43.2%) and morphological, both single and multiple, in 143 of cases (56.8%). In most of the cases (65%) these anomalies have been assessed by ultrasound scan, while in 35% by cariotype analysis. CONCLUSION: After the legalisation in 1978, cases of abortion have constantly increased. More detailed data would be helpful to better understand and face this event.

Hormonal contraception: state of the art and future perspectives.

Attempts at controlling female fertility through the utilisation of hormonal extracts date back to the beginning of the 20th century, however, practical applications in the human species were only possible when Gregory Pincus had the excellent idea of mimicking the effect of progesterone in blocking ovulation and, through this mechanism, inhibit fertility. It is important to notice that, over its 50 years of history, the original method for hormonal contraception (HC), universally known as ''the pill'', developed into a variety of modalities that today utilise a number of new routes of administration. HCs of today are exclusively made up of either oestrogen-progestin combinations or progestins alone. This does not mean that no attempt has been made to use other classes of steroids for contraception. The best known compounds tested are the so-called ''antiprogestins'', more precisely called selective progesterone receptor modulators (SPRM). Hormonal contraceptives, originally administered in the form of daily pills, can be utilised today through seven different routes: intramuscularly, intranasally, intrauterus, intravaginally, orally, subcutaneously, and transdermally. New strategies in the field of oral contraception include further dose reduction, the synthesis of new active molecules and new administration schedules. Newly marketed contraceptive rings to be inserted in the vagina, offer a novel approach by providing a sustained release of steroids and low failure rates. The transdermal route is now well established, as a contraceptive patch, a spray or a gel. The intramuscular route has also seen new products with the marketing of improved monthly injectable preparations, containing an oestrogen and a progestin. A new minipill (progestin-only preparation) containing desogestrel has been recently marketed in a number of countries and is capable of inhibiting ovulation in most women. After the first device capable of delivering progesterone directly into the uterus was withdrawn, a new system releasing locally 20 microg over 5 years, is today widely marketed with excellent contraceptive and therapeutic performance. Finally, several subcutaneously implanted systems have been also developed in the form of contraceptive ''rods'', where the polymeric matrix is mixed with the steroid and ''capsules'', made of a hollow polymer tube filled with free steroid crystals. Last, but not least, work is in progress to provide effective emergency contraception after an unprotected intercourse. Very promising in this area is the use of SPRM.

[Intrauterine death: experience at a tertiary centre of Italy]. / La morte intrauterina: esperienza in un centro terziario.

AIM: In order to analyse causes of stillbirths, we collected all the cases observed from January 1993 to December 2006 at the Department of Gynecological Sciences, Perinatology and Child Care, University ''La Sapienza'', Rome, Italy. METHODS: For each case, age of the patient, parity, country of origin, gestational age at the moment of stillbirth, clinical condition before pregnancy, pathologies occurred during pregnancy, possible therapies and autopsy of the fetus, have been collected. To evaluate and classify the obtained data, both the NICE (Neonatal and Intrauterine Death Classification according to Etiology) and the ReCoDe (Relevant Condition at Death) classifications have been utilised; the first one being more suitable than the second for our case series. RESULTS: Results showed that among 25892 labours, 186 were intrauterine deaths (7.2%). In 1999 we noticed a decrease in the number of labours of approx. 30%, due to a reduction in the number of inpatients available spaces. The number of stillbirths presented a slithering line until 2001, while after then a marked decrease has been observed. CONCLUSION: A high percentage of stillbirths had to be classified as ''unknown causes'' (26.9%). Additional prospective research, in order to achieve a better classification, is needed. All the new cases, should be classified using the most appropriate parameter, drawing attention to all the possible issues, and centralizing the data acquired.

[Infertility: a global perspective]. / Infertilitá: una visione globale.

Infertility has always existed; however, today it has a different meaning because the possibility exists, not only to treat it in some three quarters of all cases, but also, in a number of instances, to prevent it. At the same time, this improved scenario created a number of important new issues concerning public health policies and the social impact of infertility; these issues can be summarized with two words: equity and ethics. Indeed, there are intolerable differences in access to infertility care, depending on the social-economic status, as well as the country in which a couple lives. It is generally believed that, overall, in the general population a persistent form of infertility affects some 7-8% of all couples; in Europe the prevalence of infertility has been estimated at around 14%. There are important regional differences in the incidence and causes of sterility. Whereas the best known factor in western countries is an increase in the age in which women attempt to conceive, in Africa, the most important cause is represented by sexually transmitted infections. Over the last fifty years there have been major break-throws in the diagnosis and treatment of infertility, although it must be stressed that the importance and validity of therapy must be in all cases evaluated against expectant management. This is because in couples without clear signs of functional or organic pathology, conception will occur spontaneously in between 80 and 90% of the cases. At the same time, if attempts to conceive fail over a period of three years, even in women 30 years or younger, the probability of pregnancy decreases to about 40%. Finally, it must be stressed that advances in assisted reproductive technology have created not only important successes, but also significant ethical problems: on the one hand, the debate is open on the limits of artificial interventions in the field of reproduction; on the other the possibility exists that a partner may ''impose'' an infertility on the other.

[Emergency contraception]. / La contraccezione d'emergenza.

A specific formulation has been approved for use in Italy for emergency contraception (EC) in 2000. As expected, marketing of this levonorgestrel (LNG) only formulation has been accompanied by an increased interest and, often, controversies leading to even strong opposition on the part of those ethically opposed to the use of any method that may act after fertilization. At present, several trials on the exact mechanism of action and safety have been conducted, giving good reason for simplifying access, providing it free or over the counter, in several European countries. EC, also known as ''the morning after pill'' or postcoital contraception, is a modality of preventing the establishment of an unwanted pregnancy after unprotected intercourse and thus, probably, of reducing the number of voluntary pregnancy terminations. Two different forms are available: the hormonal and the intrauterine. Hormonal estrogen only EC was first proposed in the 60s and in 1974 Yuzpe following his studies proposed for the first time his combined regimen, that showed better efficacy and lower side effects. More recently, a new regimen, consisting of LNG, administered alone at the dose of 1.5 mg, was introduced and found in clinical trials to be more effective than the Yuzpe regimen, if taken as early as possible, within 72 h, thereby replacing the latter in common use. Mechanism of action of both hormonal preparations used for EC is inhibiting or delaying ovulation, therefore a prefertilization action. No effect has been reported on the process of implantation nor on an ongoing pregnancy. The WHO have developed a third regimen based on the use of the selective progesterone receptor modulator (antiprogestin) Mifepristone and conducted trials with different dosages, reporting similar efficacy and safety compared to LNG. Intrauterine EC was first proposed by Lippes in 1976. It has the advantage of being effective if inserted within 5 days after unprotected intercourse and the disadvantage of a greater complexity. In addition, this modality is truly interceptive, acting by preventing implantation. Pregnancy rates reported following EC using a device >300 mm2 of copper are consistently low (0.1-0.2%).

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials.

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Prevention and treatment of experimental breast cancer with the combination of a new selective estrogen receptor modulator, arzoxifene, and a new rexinoid, LG 100268.

The selective estrogen receptor modulator arzoxifene and the rexinoid LG 100268 were active not only as single agents for prevention and treatment of breast cancer in the rat model that uses nitrosomethylurea as the carcinogen but also showed striking synergy, both preventively and therapeutically, in a series of six experiments with a total of 465 rats. Mechanistic studies in cell culture reported here suggest that enhancement of stromal-epithelial interactions may contribute to this synergy. The possible clinical use of the combination of arzoxifene and LG 100268 for prevention of breast cancer in women at high risk, for treatment of women in the adjuvant setting, or for treatment of end-stage disease should now be considered.

Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes.

Four new antibiotic-resistant derivatives of the broad-host-range (bhr) cloning vector pBBR1MCS have been constructed. These new plasmids have several advantages over many of the currently available bhr vectors in that: (i) they are relatively small (< 5.3 kb), (ii) they possess an extended multiple cloning site (MCS), (iii) they allow direct selection of recombinant plasmid molecules in Escherichia coli via disruption of the LacZ alpha peptide, (iv) they are mobilizable when the RK2 transfer functions are provided in trans and (v) they are compatible with IncP, IncQ and IncW group plasmids, as well as with ColE1- and P15a-based replicons.

The IgA subclass responses of human lymphocytes to B-cell activators.

The induction of IgA synthesis in normal peripheral blood lymphocytes cultured in the presence of 5 different B-cell activators was studied by immunofluorescence using monoclonal antibodies to the two subclasses. The surface phenotype of normal cells after overnight culture in the absence of mitogen showed a mean ratio IgA1:IgA2 of 2.7:1; cells with cytoplasmic IgA were very rare. Results obtained on different donors after stimulation showed considerable variation; IgA1 was the predominant subclass in Epstein-Barr virus-stimulated cultures, whereas pokeweed mitogen induced a predominantly IgA2 response; cells stimulated with Staphylococcus aureus, Branhamella catarrhalis and lipopolysaccharide and unstimulated cells showed a 1:1 ratio of the subclasses. It is concluded that in this system IgA subclass expression is a function of the activator employed.