Having thrived in Eurasia for 350,000 years Neandertals disappeared from the record around 40,000–37,000 years ago, after modern humans entered Europe. It was a complex process of population interactions that included cultural exchanges and admixture between Neandertals and dispersing groups of modern humans. In Europe Neandertals are always associated with the Mousterian while the Aurignacian is associated with modern humans only. The onset of the Aurignacian is preceded by “transitional” industries which show some similarities with the Mousterian but also contain modern tool forms. Information on these industries is often incomplete or disputed and this is true of the Uluzzian. We present the results of taphonomic, typological and technological analyses of two Uluzzian sites, Grotta La Fabbrica (Tuscany) and the newly discovered site of Colle Rotondo (Latium). Comparisons with Castelcivita and Grotta del Cavallo show that the Uluzzian is a coherent cultural unit lasting about five millennia, replaced by the Protoaurignacian before the eruption of the Campanian Ignimbrite. The lack of skeletal remains at our two sites and the controversy surrounding the stratigraphic position of modern human teeth at Cavallo makes it difficult to reach agreement about authorship of the Uluzzian, for which alternative hypotheses have been proposed. Pending the discovery of DNA or further human remains, these hypotheses can only be evaluated by archaeological arguments, i.e. evidence of continuities and discontinuities between the Uluzzian and the preceding and succeeding culture units in Italy. However, in the context of “transitional” industries with disputed dates for the arrival of modern humans in Europe, and considering the case of the Châtelperronian, an Upper Paleolithic industry made by Neandertals, typo-technology used as an indicator of hominin authorship has limited predictive value. We corroborate previous suggestions that the Middle-to-Upper Paleolithic transition occurred as steps of rapid changes and geographically uneven rates of spread. The Uluzzian is a cultural unit dated within the time interval between 45 and 40 ka B.P., the time interval during which Neandertals were replaced by a population of modern humans. It is known from a number of sites in Southern Italy : CastelcGrotta La Fabbrica  is located in the Parco Naturale della Maremma, at the base of the western slopes of Monti dell’Uccellina and 1.5 km from the Tyrrhenian Sea. Colle Rotondo  is a single layer open-air site in south-central Latium. It lies on the flat top of a hill about 65.8 m asl and 2.3 km from the Tyrrhenian sea. It is a recent discovery proving the presence of the Uluzzian in a region where it was supposed to be completely absent , 16 and 40Ar/39Ar to 45.5 ± 1.0 ka. This date marks the beginning of the Uluzzian at CavalloThe Uluzzian overlies the Mousterian at all cave sites but not at the open air site of Colle Rotondo which contains a single layer only. Based on new radiocarbon results from Cavallo, Fumane, Castelcivita and Klissoura, a Bayesian statistical approach, and dates for the underlying Mousterian levels, Douka et al.  concludeThe start of the Uluzzian at Castelcivita has not yet been established. There is, however, a radiocarbon date of 40.5–42.0 ka cal BP for the upper layer which agrees well with the stratigraphic position of the sample, below the Protoaurignacian. The youngest Uluzzian site appears to be Klissoura in Greece. Its thin Uluzzian layer is contaminated by earlier and later elements but the final date is provided by tephra of the Campanian Ignimbrite that occurs at the top of the Uluzzian layer [Two important chronostratigraphic markers signal the end of the Uluzzian: the Protoaurignacian and the Campanian Ignimbrite. The Protoaurignacian is a culture-stratigraphic unit that appears in Southwestern Europe  at about 42–41 ka. The earliest appearance in Italy is at the Riparo Mochi in Liguria between 42.7 and 41.6 ka cal BP , 9. BecaThe Uluzzian underlies the Protoaurignacian at Fumane, La Fabbrica, Castelcivita and Grotta La Cala. In southern Italy the final stages of the Protoaurignacian at Serino and Castelcivita are marked by the Campanian Ignimbrite eruption. Northwest of Campania, however, the Campanian Ignimbrite does not occur for reasons explained below. Thus the end of the Protoaurignacian in Central and Northern Italy and its relation to the Early Aurignacian are not yet determined with certainty , 21.The Campanian Ignimbrite (thereafter CI) is a major volcanic eruption that originated from the Phlegrean Fields west of Naples, in the Bay of Pozzuoli. The eruption is dated to 39.88 ± 0.12 ka . CI asheThe Campanian Ignimbrite overlies the Protoaurignacian at Castelcivita and Serino in Campania  and is dThe objectives of our paper are: (a) to date the Uluzzian of Grotta La Fabbrica on the basis of newly acquired OSL dates and to place the open-air site of Colle Rotondo in its morpho-stratigraphic context; (b) to present the results of a detailed technological, typological and taphonomic analysis of the two lithic assemblages for a more precise characterization of their features; c) to view our results in the broader context of the emergence, duration and disappearance of the Uluzzian industries in Italy.The La Fabbrica collections are housed in the Dipartimento di Civilta e Forme del Sapere, University of Pisa, under the care of Carlo Tozzi. The Colle Rotondo collection is housed in the laboratory of the Dipartimento di Studi Umanistici, University of Roma 3, under the care of Massimo Pennacchioni. Tozzi and Pennacchini are co-authors of this paper. Hence permits were not required for this study.We followed the sorting procedures used by us in the analysis of other assemblages. We select all cores, core fragments, tools, tool fragments, complete or broken flakes preserving the platform, blades and bladelets, whether complete or fragments. Some debitage byproducts such as flakes from scaled pieces, Kombewa flakes and flakes with thermal scars are also recorded.Our sorting procedures exclude from technological analysis flake fragments (broken flakes without the platform) flakes < 1 cm and chunks, Siret breaks, tested cobbles and cobble fragments. These materials are bagged by large categories and remain available for specific studies. Flakes between 1 and 2 cm are classed by raw material only but flakes from scaled pieces and bipolar flakes > 1 cm are extracted and counted in debitage tables. All other unretouched flakes preserving the platform are recorded in individual Excel files by type, raw material, platform type and measurements.The La Fabbrica lithics had square and layer indicated but no systematic catalogue number. Some pieces had numbers assigned occasionally for a particular research purpose. Thus we assigned individual numbers in sequential order to retouched pieces and cores which are individually bagged in reusable zipper bags (Minigrips) with preprinted labels.The Colle Rotondo materials had year, square and sublevel marked in ink. Many had numbers but they were not in consecutive order by sublevel, the numbering was restarted by sublevel, a possible source of confusion. Hence we numbered them using sublevel as a prefix followed by the original number. The retouched pieces and cores are individually bagged with preprinted labels and stored in plastic drawers or larger plastic bags by class. Unretouched flakes and other small items have labels with their square and layer provenience and are bagged by large categories.Our analysis of lithic assemblages is directed to the identification and description of similarities and differences in reduction sequences, in the objective of production, in the selection of blanks for tool manufacture and in the typology of formal tools.Once sorted with the procedures described above, retouched pieces  and cores are recorded by catalogue number, provenience, raw material and state of preservation in separate Excel files. Length, breadth and thickness were measured in mm by digital caliper according to the morphological axis (if a tool) or the debitage axis (if a flake or a blade). Incomplete measurements (if the artifact is broken and incomplete) are identified by a symbol in front of the number. Raw material is identified by macroscopic examination with a 10x hand-lens and comparison with known geological samples.Most analytical details  are coded and described in the We read on each core and flakes the direction and sequence of removals thus reconstructing the manufacturing steps for each piece. On cores this information is represented in a visual way that reveals the general structure of the piece. The white lines indicate the scar outline; the scar orientation is indicated by a white arrow, a white dot indicates the presence of a negative bulbs. Repeating patterns of technical features allows the definition of technical types.A dynamic understanding of each assemblage is provided by the ordering of static types into a sequence of manufacturing steps. This is the principle of chaînes opératoires which originated in the 1960s and came into common use in the 1990s . The objScaled pieces and bipolar cores are characteristic of the Uluzzian yet bipolar cores are often confused or lumped with scaled pieces . The Mousterian is dated to 44 ± 2.1 and the Uluzzian is dated to 40 ± 1.6 ka . An erosWe had originally planned to study in the same way the Protoaurignacian of which a technological and typological analysis was published in 2012  based on2, 1c) based on differences in color and the presence of discordant surfaces. The in situ material comes from approximately 15 sq m and was in part protected from erosion by large blocks fallen from the roof Bipolar cores are quite abundant  and occaDebitage  equipped with an EDAX DX4 spectrometer for qualitative chemical elemental analysis. Chemical analysis of hafting adhesives. Two of us (ID and JJL) applied gas chromatography/mass spectrometry (GC/MS) to the characterization of amorphous materials on three samples: two from La Fabbrica and one from Colle Rotondo. The sediment corresponding to the sampled pieces was also analyzed to rule out external contamination. Full analytical details are provided in the The three samples had clearly visible residues on a lateral edge. They are 1) an unretouched flake from Colle Rotondo, 2) one side scraper from the Mousterian layer 1b of La Fabbrica and 3) one end scraper from the Uluzzian layer of La Fabbrica . MicrosaPinaceae. The profile of the lipid components suggests the co-presence of plant and animal fats, due to the occurrence of fatty acids with even and odd carbon number, ranging from C12 (dodecanoic acid) to C25 (pentacosanoic acid) [The Uluzzian end scraper instead gave significant results . The chric acid) .Hafting adhesives have been documented by chemical analysis such as GC/MS in Middle Paleolithic sites, such as birch bark pitch at the site of Campitello Quarry in Tuscany  dated to the end of MIS 7 (about 200 ka) and at the German site of Königsaue A with an estimated age of 80,000 years . At Umm Colle Rotondo in Latium is a recent discovery proving the presence of the Uluzzian in a region where it was supposed to be completely absent –16. It wThe site is located in southern Latium, 8 km north of Anzio . It liesThe area excavated in 2011 and 2012 by one of us (Massimo Pennacchioni) is about 6 sq m. . The excMore than 80% of all artifacts come from the first three excavation levels . The lowest two excavation levels (levels 4–5) were found only in one square. The most common raw material is flint.On a random sample of 100 artifacts, 23% are slightly patinated, often covered with reddish-brown concretions and black manganese stains. Patination, a form of weathering where sunlight and water play a certain role  might suThere were very few badly altered bone fragments and no charcoal. One small Levallois core  and one small Levallois flakes were found in square K10 and L10 respectively Fig E in . The corThe stratigraphic features of the terrains exposed by archaeological excavation at Colle Rotondo are shown in Figs The thin section of sample CR1  shows thThe Colle Rotondo site is located on the flat surface of an E-W elongated hill, bordered by the steep flanks of two convergent streams . The canThe relic paleo-surface of Colle Rotondo was mapped by Marra et al.  who recoGeochronological constraint provided by the 40Ar/39Ar age of 129±1 ka yielded by a tephra layer intercalated within the sedimentary succession forming the coastal terrace at ca. 36 m a.s.l. allowed Marra et al.  to correTherefore, the Colle Rotondo paleo-surface originated circa 240 ka (age of the MIS 7.5 highstand) above the sandy deposits of an alluvial to back-beach coastal plain, and since then experienced repeated cycles of very limited erosion (during the sea-level lowstands) and eolian/colluvial deposition (mainly during the highstands). In contrast, the present day deep fluvial incisions progressively developed since the MIS 6 lowstand, and cut deeper and deeper the original morphology due to the intervening uplift that raised the coastal area for an amount of 50 m, during the last 200 ka.Since about 60,000 years ago, corresponding to a time when the sea-level fell down to -100 m  the CollThis fact implies that the lithic material was brought in situ by man after 60,000, or that it represents relic material which was deposited on the surface after its formation 240 ka, and that it experienced only limited colluvial/runoff transport. In fact, the artifacts, with diameter ranging from 2 mm to 9 cm, are incorporated within a 6 to 20 cm thick silty sand layer  and at the top  of the layer incorporating the artifacts yielded unexpected young ages ranging from 18,180 ± 950 for CR3, 15,870 ± 1,100 for CR 2 and 14,640 ±960 for CR1  since the Last Glacial termination (ca. 14 ka). This picture is consistent with the overall climatic condition during the last 60 ka, characterized by cold and arid conditions during all the full glacial period 60 through 18 ka, favoring eluviation processes rather than eolian deposition and soil formation, followed by warm and humid condition in the Holocene, accounting for the rapid accumulation of an eolian soil above the former ground surface.The lithic assemblage, unaffected by eolian transportation, is likely to have remained close to the exposed surface until around 14 ka, and then rapidly buried: a fact that fully justifies the OSL dates yielded by the sediment immediately below the artifacts  and immediately above them .Thus the quoted OSL ages do not refer to the depositional event but to a later sun exposure: specifically the last time of exposure before rapid burial of the archaeological layer.The assemblage remains undated but the similarity to the La Fabbrica assemblage . Its prAt both sites bipolar blades and bladelets are few and do not have retouch, suggesting that their production was occasional. At Colle Rotondo blades and bladelets by direct percussion are fewer than at La Fabbrica but some were retouched, including 2 lunates and 2 truncations. The lack of dedicated bladelet cores suggests that bladelet production was just part of knapping sequences of unidirectional cores. Blades may have been produced during the initial knapping phases of unidirectional cores, as suggested by the fact that three of the five blades are cortical or partly cortical.Dedicated bladelet cores are thus a difference between Colle Rotondo and La Fabbrica. At La Fabbrica there are two bladelet cores made by direct percussion and three made with the bipolar technique. However blades and bladelets have only some partial marginal retouch. In conclusion, at both sites blades and bladelets, whether by direct percussion or by the bipolar technique, represent only a minor component of the assemblage.The bipolar technique appears to be more frequent at La Fabbrica  have been described in the Castelcivita Uluzzian. They come from near the top of the Uluzzian layer and are interpreted by  as precuEight Dufour bladelets in the Uluzzian of Cavallo Cave are considered by  as evideWe have not done detailed comparisons with the Uluzzian layers of Grotta del Cavallo (E and D) described and abundantly illustrated by Palma di Cesnola –62 for tThe ongoing controversy about the stratigraphic position of two modern human teeth attributed to the Uluzzian layers , 63, 64 This is clear evidence that the Uluzzian is a coherent and valid culture-stratigraphic unit with an approximate duration of 5 millennia. We find difficult to fit in this picture the Uluzzian assemblages from Fumane Cave of layers A4 and A3 . The LevThree alternative hypotheses have been proposed for the authorship of the Uluzzian: that it is the product of modern humans dispersing into Eurasia from the African continent –4, that The late age of the Klissoura assemblage would seem to exclude a migration of populations from Greece into Italy . We agreWe can look at evidence of continuities or discontinuities between the Uluzzian and the preceding and succeeding culture units in Italy. We are aware of the fact that techno-typology or stratigraphic continuity/discontinuity are not necessarily a predictor of hominin taxonomy. But most work in science is purely observational and we expect that the implications of the patterns we describe will require some time to fall into place and may provide some insights into a theory of cultural transmission and relatedness which in prehistory is at the elementary level.The Uluzzian occupation is sometime, but not always, separated from the Mousterian by a hiatus in the stratigraphic sequence. At Castelcivita the stratigraphic contact between the Mousterian and the Uluzzian is marked by a clear sedimentological difference indicating a hiatus (of unknown length) . At Grot14C dates partly overlap [At Fumane on the contrary there is no separation between the last Mousterian layer (A5) and the first Uluzzian layer (A4). The  overlap  and in s overlap . At Grot overlap .According to several authors , 15, 37 High frequencies of side scrapers and denticulates in the Uluzzian of Cavallo and Castelcivita  have beeA debitage oriented to the production of flakes is certainly not a characteristics of the Early Upper Paleolithic. To find a Western European industry with an abundance of flakes used as tools blanks we have to wait about 20,000 years for the Badegoulian which is dated between 22 and 20 ka calibrated BP . HoweverUnprepared cores with short reduction sequences are a characteristic feature of the Uluzzian. This kind of cores occurs throughout the Paleolithic but they are a common component of the Middle Paleolithic and are found also in the Châtelperronian . This kiThe general view seems that elements such as very high frequencies of scaled pieces, bone tools, bladelets, lunates, use of ochre and a few marine shell ornaments  are seen as innovations that distinguish and separate the Uluzzian from the Mousterian. The significance of these components is discussed below.Scaled pieces (the French term is pièces esquillées) are not indicators of specific industries. As is often the case with stone artifacts their significance depends on frequencies. Scaled pieces occur in the Developed Oldowan of Middle Bed II at Olduvai, in the Acheulian site of TK in Upper Bed II and in other Olduvai localities up to the Masek Beds  but hey Compared to the Mousterian we do observe a strong increase in the number of scaled pieces in the Uluzzian of Castelcivita, Cavallo, La Fabbrica and Colle Rotondo , in the In general, Italian Mousterian assemblages contain scaled pieces in low frequencies. In the Mousterian of Castelcivita scaled pieces are 2.2% (10 of 444) (see p. 101 in ). There Archaeological evidence shows that personal ornaments such as perforated and ochred marine shells are documented in the Mousterian at Cueva de los Aviones and Cueva Anton in Spain –84 and aFragments of non-local hematite have been found at Maastricht-Belvédère Site C, dated to minimally MIS 7 (250–200 ka). Lumps of ochre and ochre staining on stalagmite containers have been found also at other sites such as Amsterade-Allée (Netherlands) with an estimated date 75,000 BP, Les Bossats (France) dated to 47,000 y ago and Ciaorei Cave in Romania, proving that the use of red ochre was a component of Neandertal cultural behavior –96. SeveThe idea that backed bladelets provided modern humans with projectile weaponry that gave them a huge advantage on Neandertals equipped only with hand-cast spears, once modern humans moved out of Africa, was suggested a few years ago by Brown et al . SupportFormal bone tools made with techniques specifically conceived for bone materials such as splitting and wedging, scraping and polishing are common in Upper Paleolithic sites but some also occur in the Mousterian. Four lissoirs (a French term meaning “smoothers”) described by  come froIn the Châtelperronian of Grotte du Renne 48 bone awls made by scraping and some marked with notches have been studied by . Almost It could be argued that many of these apparent novelties were just episodically present in Neandertal assemblages and only later became a stable component of cultural behavior and that it is in the Uluzzian that these novelties appear to be stable elements. However this review makes it clear that the question of hominin authorship cannot be answered by an archaeological approach to patterns of continuity or discontinuity because in the temporal context of “transitional industries” technology and typology are in fact controlled by too many variables. Innovations and adoptions of new technologies are not the exclusive behavior of one hominin species, as exemplified by the Châtelperronian, defined as a fully Upper Paleolithic industry with blade and bladelet production and bone-tool production yet made by Neandertals , 103–110The replacement of the Mousterian by the Uluzzian with all its defining elements and the replacement of the Uluzzian by the Protoaurignacian appear rapid with no evidence of a gradual change. A similar abrupt replacement of the last Mousterian level by the Protoaurignacian has been observed at Grotte Mandrin  where a very short time, in the order of a few seasons or a few years, separates the two occupations. This is documented by a precise occupation sequence established by using sooted concretions and clasts in stratigraphic order . This suIf we consider that the Protoaurignacian arrived in southwest Europe, including Italy, between 42,000–41,000 , 114 thiAn alternative hypothesis is that the “innovative” elements such as blades, bladelets, bone tools and ornaments would indicate modern culture influence through stimulus diffusion (i.e. acculturation)  since thThis research has allowed us: (a) to present detailed technological and typological analyses of the assemblages from Grotta La Fabbrica and from the new site of Colle Rotondo with particular attention to each site age, chronostratigraphic context and taphonomy (b) to show that the general technological and typological features of the assemblages of Castelcivita, Grotta del Cavallo, La Fabbrica and Colle Rotondo define the Uluzzian as a valid cultural unit with an approximate duration of five millennia. The Uluzzian was replaced by the Protoaurignacian about one or short of two millennia before the eruption of the Campanian Ignimbrite; (c) to show that the question of who were the makers of the Uluzzian cannot be answered by an archaeological approach to patterns of stratigraphic and techno-typological continuity or discontinuity. In the specific context of transitional industries, with disputed dates about the arrival of modern humans in Western Europe and assemblages from central and southeastern Europe only partly known, technology and typology cannot provide conclusive evidence of hominin taxonomy and there is ongoing controversy about the stratigraphic position of two modern human teeth attributed to the Uluzzian layers of Grotta del Cavallo. The answer will have to come from DNA evidence and/or more human remains. DNA analysis of La Fabbrica sediment is being planned. (d) Our work confirms previous suggestions  that theS1 File(PDF)Click here for additional data file.S2 File(DOCX)Click here for additional data file.S3 File(PDF)Click here for additional data file.S4 File(DOCX)Click here for additional data file.S5 File(PDF)Click here for additional data file.


P < 0.01). Based on Haploview software analysis, the haplotype block Ars3208008 Grs41309931 Grs2297441 Ars1291207 exhibited the highest frequency, but, haplotype blocks Ars3208008 Trs41309931 Grs2297441 Ars1291207 and Crs3208008 Grs41309931 Grs2297441 Ars1291207 were associated with breast IDC risk. This study also detected associations between DcR3 gene polymorphisms and the clinicopathological features of breast IDC, including lymph node metastasis and C-erbB2, P53, estrogen receptor and progesterone receptor status. These data indicate that DcR3 gene polymorphisms are associated with sporadic breast IDC risk in Northeast Chinese females.Decoy Receptor 3 (DcR3), also called TNFRSF6β, is a member of the tumor necrosis factor receptor superfamily and is a soluble receptor for FasL. DcR3 is overexpressed in cancers and contributes to tumorigenesis through immune suppression and promotion of angiogenesis. We found that DcR3 is overexpressed in breast infiltrating ductal carcinoma (IDC) cells as compared with normal controls. We also conducted a case-control study analyzing associations of DcR3 polymorphisms with breast IDC risk. Subjects included 531 females with breast IDC and 592 age-matched healthy controls. Four DcR3 single nucleotide polymorphism loci with minor frequencies of more than 5%  were genotyped using polymerase chain reaction restriction fragment length polymorphism and sequencing. Our results revealed significant differences in rs41309931genotypes and alleles ( Decoy Receptor 3 (DcR3), which maps to chromosomal region 20q13.3, is also called tumor necrosis factor receptor super family member 6β (TNFRSF6β) and can bind FasL, LIGHT and TL1A . DcR3 caInfiltrating ductal carcinoma (IDC) is the most common type of breast cancer and accounts for about 80% of cases . Both naSingle nucleotide polymorphisms (SNPs), as natural sequence variations, may lead to differential DcR3 expression. With respect to genetic variation, SNPs have been utilized in breast cancer research , 17. To P = 0.0001). DcR3 expression was positive in 76.4% of tumors and 40.0% of pericarcinomatous tissues (Supplementary File A). Thirty breast IDC tissue samples exhibited positive ER, PR and HER2  expression. There was no significant relationship between DcR3 and ER positivity in the tumor samples, and we also found the similar results for PR and HER2 . rs41309931 in the 5′-near gene region  was associated with breast IDC. In the allele analysis, the T allele in rs41309931 (P = 0.001) had lower frequencies in tumors. We corrected the P-value in this allele for multiple testing by using 10,000 permutations in the Haploview program (P = 0.0033).Genotype and allele frequencies are shown in Tables P = 0.0053) and CGGA  had a higher frequency in cases (P = 0.0061). The other haplotypes were not significant (P > 0.05).Haploview 4.1 analysis produced seven haplotypes with frequencies > 5% Table . The mosP = 0.030). The AG genotype in rs2297441 had a lower frequency in C-erbB2 positive cases (P = 0.033). In rs2297441, the AG and AA genotypes  had lower frequencies in P53 positive cases and AA (P = 0.044) had a higher frequency in ER positive cases.We analyzed the relationships between DcR3 polymorphisms and a series of breast IDC clinicopathological features, including tumor size, lymph node metastasis and the statuses of ER, PR, C-erbB2 and P53. No DcR3 polymorphism was associated with tumor size. However, the frequencies of the AA genotype and A allele in rs2297441 were higher in lymph node metastasis positive cases . The A allele in rs2297441 also had a higher frequency in lymph node metastasis, ER and PR positive cases , but had a lower frequency in P53 and C-erbB2 positive cases (P = 0.001 and 0.015). After correcting the P-value for multiple testing, we found that only the A allele in rs2297441 was significantly associated with P53 and C-erbB2 positivity .We found that the T allele in rs41309931 had a higher frequency in ER and PR positive cases (P = 0.0309 and 0.0191). Haplotype AGGG  (7.7%) had a lower frequency in C-erbB2 and ER positive cases (P = 0.0088 and 0.0483). CTGA  had a lower frequency in PR positive cases. After correcting for multiple testing, only the AGGG haplotype in lymph node metastasis positive cases was significant (P = 0.0467), and no associations were observed between haplotypes and P53, C-erbB2, ER or PR status (P > 0.05).We also analyzed associations between the haplotypes and clinical features. Haplotype AGGG  (7.7%) had a higher frequency in lymph node metastasis and P53 positive cases  exhibited the highest frequency of all haplotypes in the DcR3 gene, but was not significant. Haplotype ATGA  had a lower frequency in cases than controls and could thus be protective against breast IDC. Haplotype CGGA  had a higher frequency in cases than controls.Steroid hormone receptors are valuable for predicting breast cancer prognosis and are regarded as predictive markers of endocrine therapy outcome . Our resIn previous research, DcR3 was thought to be as a possible cancer biomarker , but fewU test was used to analyze differences in DcR3 expression between tumor and pericarcinomatous samples.We collected 55 carcinoma and pericarcinomatous tissue samples from female breast infiltrating ductal carcinoma (IDC) patients with clear pathologic diagnoses. Paraffin-embedded samples were stained with purified mouse anti-human DcR3 primary IgG antibody (BioLegend) followed by the ImmunoCruz mouse LSAB Staining System . Images were screened using a Nikon Eclipse 80i Microscope. For each specimen, immunohistochemical (IHC) staining proportion and intensity scores were determined . ImmunosThree breast cancer cell lines, including MD-MBA-231, SKBR3 and T47D, along with human mammary epithelial cells (HMEC), were sourced from the breast cancer tissue bank of the Third Affiliated Hospital of Harbin Medical University. All samples were resuspended in lysis buffer and 2 mM PMSF . DcR3 expression was detected by immunoblot using β-actin as a housekeeping protein. Primary antibodies were purified mouse anti-human DcR3 IgG  and mouse anti-human β-actin monoclonal antibody . The secondary antibody was peroxidase-conjugated Affinipure goat anti-mouse IgG . The Luminata Forte Western HRP Substrate (Milliipore) was applied in exposure.This study included 531 female breast IDC cases (average age 49.5 ± 9.9 years) and 592 normal controls (average age 45.4 ± 9.9 years). All breast IDC cases, which were definitively diagnosed histopathologically in 2009, were recruited from the Breast Surgery DNA bank of the Third Affiliated Hospital of Harbin Medical University. Clinical feature information included tumor size, lymph node metastasis, human epidermal growth factor receptor 2 (C-erbB2), estrogen receptor (ER), progesterone receptor (PR) and tumor protein 53 (P53) statuses (Table hapmap.ncbi.nlm.nih.gov/) and dbSNP databases (www.ncbi.nlm.nih.gov/projects/SNP/). We chose six DcR3 SNPs, including rs3208008 and rs41309931 in the 5′-near gene region, rs2297441 in the 5′-UTR, rs2257440 and rs909341 in exons and rs1291207 in an intron. The minor genotype frequency of the six SNPs was more than 5% in the CHB population.DcR3 gene SNPs were selected using the Hapmap (2+ plus), 4 μl dNTPs, 5 U TaqDNA polymerase  and 0.5 μl of each primer . Primers and restriction enzymes for PCR-RFLP genotyping are shown in Table Whole blood genomic DNA was extracted using the Universal Genomic DNA Extraction Kit Ver. 3.0 . All six SNPs were genotyped via polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) assay. The polymorphic region was amplified by PCR using a T-Gradient Thermoblock PCR System  in a 50 μl reaction solution containing 0.5 μg genomic DNA, 5 μl 10 × PCR buffer  and 95% confidence interval (CI). Disease characteristics were compared in all patients using the chi-square test. Genotype frequencies determined using different models of inheritance  were analyzed using the chi-square test. Allele, genotype and haplotype frequency distributions were compared using the chi-square test, with P < 0.05 representing statistical significance. Statistical analyses were performed using SPSS 16.0 software.Genotype frequencies of all SNPs were determined for Hardy-Weinberg equilibrium (HWE) assessment. We selected four SNPs for which enough data was available to perform statistical analyses. We used Haploview 4.1 software to tag all common haplotypes and their frequencies in both breast IDC cases and controls. To correct for multiple testing bias, we ran 10,000 permutations to determine According to our limited case–control study results, we found that DcR3 SNPs differ between breast IDC cases and controls in a Chinese population. We also investigated the associations between DcR3 gene polymorphisms and breast IDC clinical features, such as lymph node metastasis and the statuses of ER, PR, CerbB-2 and P53. We can conclude that DcR3 SNPs may be associated with breast IDC risk in females of Northeast China.


Francisella tularensis, and the structural characterization of three crystal forms which grow under similar conditions. One crystal form reveals a compressed state of the ClpP tetradecamer and two forms an open state. A comparison of the two types of structure infers that differences at the enzyme active site result from a conformational change involving a highly localized disorder‐order transition of a β‐strand α‐helix combination. This transition occurs at a subunit‐subunit interface. Our study may now underpin future efforts in a structure‐based approach to target ClpP for inhibitor or activator development. Proteins 2016; 85:188–194. © 2016 Wiley Periodicals, Inc.Caseinolytic proteases are large oligomeric assemblies responsible for maintaining protein homeostasis in bacteria and in so doing influence a wide range of biological processes. The functional assembly involves three chaperones together with the oligomeric caseinolytic protease catalytic subunit P (ClpP). This protease represents a potential target for therapeutic intervention in pathogenic bacteria. Here, we detail an efficient protocol for production of recombinant ClpP from  AAAATPases Associated with diverse cellular ActivitiesASAaccessible surface areaThe ATP‐dependent caseinolytic proteases (Clp) maintain protein homeostasis in bacteria, especially under conditions of stress.+ (ATPases Associated with diverse cellular Activities) chaperones termed ClpA, ClpX and ClpC, that regulate the presentation of substrates into the active sites.Bacillus subtilis (BsClpP),Mycobacterium tuberculosis,Staphylococcus aureus.The functional Clp protease is a large multi‐protein complex, with an assembly of 14 subunits of the promiscuous serine protease ClpP at the core. This ClpP tetradecamer, a dimer of heptamers each arranged in a ring, displays an overall cylindrical shape forming an internal chamber where proteolysis of unfolded proteins occurs. The catalytic core is surrounded by accessory proteins, AAAS. aureusListera monocytogenes.Francisella tularensis, based on similar considerations applied to targets in Pseudomonas aeruginosa.F. tularensis is a highly virulent Gram‐negative aerobe classified as a category (A) bio‐warfare agent by the Centers for Disease Control and Prevention (http://www.cdc.gov). We initiated a study of F. tularensis ClpP (FtClpP) to lay the foundation for early stage drug discoveryFtClpP, in an open conformation at a resolution of 2.3 Å, was available in the Protein Data Bank (code: 3P2L) but there is no associated publication and only limited experimental details are available. Here we present the efficient production of a recombinant source of FtClpP, the crystal structure of a compressed form of the enzyme at 2.8 Å resolution and two crystal forms that reveal the structure of an open state at 1.9 and 1.7 Å resolution.Clp proteases have been investigated as antibacterial drug targets and some inhibitors do appear to attenuate the virulence of FtClpP:I 5G1Q, FtClpP:II 5G1R and FtClpP:III 5G1S.PDB codes: Escherichia coli K12, encoding FtClpP (UniProt: Q5NH47) was purchased (GeneWiz). This gene was cloned into a modified pET15b vector (Novagen) that adds a hexahistidine tag (His6‐tag) followed by a TEV (tobacco etch virus) protease cleavage site at the N‐terminus of the product. The restriction enzymes used were BamHI (at the 5' end) and NdeI (at the 3' end of the DNA strand). The construct was sequenced as a check. Recombinant expression was carried out in E. coli BL21(DE3). The bacteria were cultured in 1 L of LB broth containing 50 μg mL−1 carbenicillin at 37°C until an O.D. of 0.6 at λ = 600 nm was achieved then the temperature was decreased to 20°C. Gene expression was induced by adding 1 mM IPTG and continued overnight. Cultures were centrifuged  to harvest the cells. The sample was then resuspended in buffer A (20 mM Tris‐HCl pH 7.4 and 200 mM NaCl) containing an EDTA‐free protease inhibitor cocktail tablet (Roche). Cell disruption was carried out using a French press (20 psi) followed by centrifugation . The soluble proteins were loaded on a 5 mL HisTrap HP column , loaded with Ni2+, to perform affinity chromatography. The buffers employed for FtClpP purification were buffer A and buffer B (buffer A with 0.8M imidazole). A fraction at 20% buffer B contained the desired product FtClpP. The sample was incubated with His6‐tagged TEV protease (1 mg per 20 mg protein) at 4°C overnight while performing dialysis into buffer A to remove the imidazole. A second affinity purification step was then performed under the same conditions but this time to separate the His6‐tag and TEV protease from the desired product. Size exclusion chromatography was then pursued with FtClpP with a HiLoad 16/60 superdex 200 prep grade column  and buffer A. The yield was 10 mg FtClpP per litre of bacterial culture and a high level of protein purity was confirmed using SDS‐PAGE and MALDI‐TOF‐MS analyses. Protein concentration was determined using the predicted ε(FtClpP) = 8940 M−1 cm−1 calculated with PROTPARAM.Synthetic DNA, codon optimized for expression in FtClpP samples were concentrated to 20 mg mL−1 and various commercially available crystallization conditions were tested using 1:1 and 1:2 protein solution to reservoir ratios  in sitting drop 96‐well plates. Orthorhombic crystals were obtained from The Classics Suite (Molecular Dimensions) condition B4  and 0.1M sodium acetate pH 4.6) and optimized using 1:1 protein solution to reservoir ratio in a final drop volume of 4 μL with the hanging drop method.P21212 and unit cell dimensions of a = 113.5, b = 125.9, c = 96.95 Å. The Matthews coefficient calculated with the CCP4i suiteVm 2.2 Å3 Da−1 and bulk solvent about 45%). The FtClpP structure with PDB code 3P2L , also with seven subunits in the asymmetric unit, provided a model of a single subunit, without residues 128–149, for molecular replacement calculations in PhaserMR.FtCLpP:I and represents the closed or compressed form of the tetradecamer assembly. Diffraction data were also collected at ESRF beamline BM30‐A from two samples, both of which displayed low mosaic spread (0.2°). High‐resolution data were processed and analyzed using XDSP21212 or an alternative setting) but two distinct forms are evident with unit cell dimensions of a = 117.90, b = 128.20, c = 98.26 Å and a = 98.49, b = 128.23, c = 355.18. These structures are named FtClpP:II and FtClpP:III and represent open forms of the protein assembly. The Matthews coefficients (Vm of 2.4 Å3 Da−1 and bulk solvent about 50%) suggested the presence of 7 and 21 subunits in the asymmetric units in FtClpP:II and FtClpP:III, respectively. FtClpP:II was solved using PhaserMR with subunit A from the 3P2L model in a similar fashion as employed for FtClpP:I. In the case of FtClpP:III, the search model was the refined FtClpP:II heptamer. The general refinement procedure that was applied in all three cases started with a round of rigid body refinement using REFMACFtClpP:I was terminated when we were satisfied that the reliable parts of the polypeptide had been modeled and that disordered residues were excluded. No attempts were made to include solvent in the refinement given the modest resolution. In the case of the high resolution FtClpP:II and III structures the refinement extended to including rotamers, water molecules and ligands. Tight non‐crystallographic symmetry restraints were used at the beginning of all refinements then removed gradually. The refinements were ended when inspection of the maps suggested no more changes were justified, and no significant changes in the Rfree and Rwork were observed. Crystallographic statistics are presented in Table Crystals were flash‐cooled directly from drops and diffracted to 2.8 Å resolution in‐house. These samples were stored for use at the synchrotron. In one case, data were collected in‐house using a Rigaku M007HF X‐ray generator with a Saturn 944HG+ CCD detector, a 10° θ offset to compensate for a large mosaic spread of 0.9°, 10 seconds of exposure per 0.5° oscillation covering 115°. Data integration and analysis were carried out using iMOSFLMB‐factors of each subunit were determined using the program BAVERAGE.www.pymol.org) and ALINE.The models were analyzed using MolProbity.E. coli expression system and a robust purification protocol for FtClpP was established that yielded about 10 mg of enzyme per liter of bacterial culture. Analysis by SDS‐PAGE and mass spectrometry (data not shown) indicated that the samples were of high purity and of the correct mass. Size exclusion chromatography, in the presence and absence of 10% glycerol to assist solubility indicated a high level of purity and an oligomer, a single species of approximate mass 290 kDa . Subsequently when the sample was applied to a native gel, two species become evident with approximate masses of 160 kDa and 290 kDa . This suggests the presence of heptameric and tetradodecameric species in the material used for crystallization experiments. Access to sufficient quantities of material supported a crystallization screen and led to the identification of reproducible crystallization conditions. Three crystal forms of FtClpP have been characterized and these correspond to a closed or compressed conformation (termed FtClpP:I) and two structures of an open state (FtClpP:II and FtClpP:III), determined at 2.8 Å, 1.9 Å and 1.7 Å resolution, respectively. The structures were solved by molecular replacement  in the UniProt database (http://www.uniprot.org/) revealed 405 reviewed/curated entries. These amino acid sequences were aligned and displayed a range of 40–70% identity with FtClpP extending to strict conservation of the three catalytic residues. Strong conservation of amino acid types was noted in elements of secondary structure with the exception of the C‐terminal region of α5. These features are evident in the amino acid alignment of FtClpP with three orthologues for which structures have been reported  as depicted in Supporting Information Figure S2. The sequence identity between FtClpP and BsClpP is 45% and about 60% with EcClpP and MtClpP. Extending from this level of sequence conservation we note a high level of structural similarity is maintained between FtClpP and BsClpP (PDB code: 3MT6), EcClpP (PDB code: 3TT7) and MtClpP (PDB code: 5DZK) which becomes evident from the least‐squares overlay of all Cα positions in the open form asymmetric units. The r.m.s.d. values are 0.5 ± 0.1, 0.7 ± 0.1, 1.3 ± 0.1 Å, respectively.A search for bacterial ClpP sequences  and two loops  with α‐helices  of an adjacent subunit. Two heptamers associate to create the tetradecamer exploiting primarily hydrophobic interactions between two α5 helices that are aligned antiparallel to each other, and with hydrogen bonds between the main chain functional groups on a pairing of β9 strands [Fig. FtClpP:I and II structures, a heptamer constitutes the asymmetric unit and the physiologically relevant tetradecamer is created across a crystallographic two‐fold axis of symmetry. In FtClpP:III there are 21 subunits in the asymmetric unit and these are arranged to form a complete tetradecamer and one heptamer, which again is positioned at a two‐fold axis that generates another tetradecamer. It is intriguing that the purified sample displayed both heptamer and tetradecamer species when analyzed in a native gel suggesting that distinct conformational states might be present and this may explain why we obtained open and compressed oligomer structures.FtClpP:II and III, the open form structures. The overlay of Cα positions in the tetradecamers gives an r.m.s.d. of 0.16 Å, so the later is primarily used for further analysis given that it has been determined to a slightly better resolution. In the case of FtClpP:III, 35 ± 2% of a subunit accessible surface area (ASA) is involved in tetradecamer formation and 43 ± 1% in FtClpP:I. The buried surface areas for the tetradecamer assemblies are approximately 70% and 55%, respectively. These values indicate the presence of stable macromolecular assembliesFtClpP:III buries a significantly greater surface area of the oligomeric assembly.There is a high similarity between FtClpP:III  indicates a high degree of similarity. The same observation applies to the FtClpP:I subunits with an average r.m.s.d. 0.21 ± 0.03 Å. Performing pairwise overlays between subunits of FtClpP:I and FtClpP:III (r.m.s.d. 0.38 ± 0.04 Å) indicate minor differences, which primarily reside in strands β1 and β2 and at the C‐terminus of the polypeptide. Even when heptamers are compared, there are no major structural differences (r.m.s.d. of 0.78 Å). In Figure FtClpP [Figs. An overlay of Cα positions of individual subunits in BsClpP there is a change driven by the distortion/straightening of α5.FtClpP we observe something different. The handle in the open form, the α5‐β9 segment, is well ordered but in the closed form we did not observe interpretable density with disorder evident. This results in a rearrangement of the subunit positions with respect to each other and in the loss of the active site groove organization [Figs. FtClpP however are similar to those previously described, for example, for BsClpP.As in other ClpP structures, it appears that the conformational change between open and closed forms is associated with an order/disorder transition of the “handle” subdomain [Fig. Supporting InformationClick here for additional data file.


Idiopathic granulomatous mastitis (IGM) is a rare chronic inflammatory disease of the breast, and its etiology remains not fully elucidated. IGM is observed more often in patients with autoimmune disease. Hyperprolactinemia is observed during pregnancy, lactation, and a history of oral contraceptive use. A 39-year-old patient with no history of oral contraceptive use presented with complaints such as redness, pain, and swelling in her left breast. Ultrasound and magnetic resonance imaging (MRI) revealed a suspicious inflamed mass lesion. Core biopsy was performed to exclude breast cancer and to further diagnose. The breast abscess was drained and steroids were given for treatment. In order to monitor any progression during the three months of treatment, hormone levels were routinely examined. Prolactin level was above the reference range, and pituitary MRI revealed a pituitary prolactinoma. After treatment with prolactin inhibitors, IGM also improved with hyperprolactinemia. This report emphasizes attention to hyperprolactinemia in cases of IGM diagnosis and treatment. IGM is a recurrent chronic inflammatory disease characterized by noncaseating granuloma, lobule inflammation, and rare breast abscess formation. Clinical and radiological features may be indistinguishable from breast cancer . The etiA thirty-nine-year-old single patient with no children and no history of oral contraceptive use was admitted to our clinic with complaints of redness, pain, and swelling in her left breast . She hadProlactin levels returned to normal and there was resolution of IGM after 4 months. Follow-up included monitoring of CRP levels. No recurrences were observed during a four-year follow-up period.In 1972, Kessler and Wolloch first defined granulomatous mastitis (GM) . Secondaα1-antitrypsin deficiency, diabetes, breast trauma, obesity, race, smoking, and infectious agents. IGM is more prevalent in the Mediterranean Region and in Asia [The etiology of IGM is not fully understood, but autoimmune and hormonal disorders are often discussed as causes , 3. IGM  in Asia . This caA close association between IGM and hyperprolactinemia has been reported , 5. AlsoIGM presents with complaints such as painful breast mass, redness, abscess, and fistula and sometimes mimics cancer because of withdrawal of the nipple and peau d'orange appearance. It is rarely accompanied with axillary lymphadenopathy . UsuallyGenerally, in the absence of secondary infection, there is no growth in abscess culture , 3.Gram stain and cultures should be performed in addition to PAS (periodic acid shift) staining for fungi and Ziehl-Neelsen stain for tuberculosis , 9. IGM Corticosteroids are the most common drugs administered for the treatment of IGM . Other dRecurrence rates can rise up to 50%. Follow-up recommendations include 3–6-month intervals for the first 2 years , 10. In IGM is a disease associated with autoimmunity and hormonal disorders. When investigating the etiology of IGM, prolactin levels should always be checked. Prolactin elevation should be monitored particularly closely and continuously and for a prolonged period of time in severe cases. Prolactin elevation is known to increase immunity and inflammation. While investigating the causes of hyperprolactinemia, the primary focus should be the presence of a pituitary adenoma. Prolactin inhibitors will improve treatment success of IGM with reducing recurrence in cases with prolactin elevation.


Internalizing problems are the most prevalent mental health problems in adolescents. Transdiagnostic programs are promising manners to treat multiple problems within the same protocol, however, there is limited research regarding the efficacy of such programs delivered as universal prevention programs in school settings. Therefore, the present study aims to investigate the efficacy of a video-based transdiagnostic rational emotive behavioral therapy (REBT) universal prevention program, for internalizing problems. The second objective of the present paper will be to investigate the subsequent mechanisms of change, namely maladaptive cognitions.1), post-intervention (T2), 3 months follow-up (T3) and 12 months follow-up (T4). Intent-to-treat analysis will be used in order to investigate significant differences between the two groups in both primary and secondary outcomes.A two-arm parallel randomized controlled trial will be conducted, with two groups: a video-based transdiagnostic REBT universal prevention program and a wait list control. Power analysis indicated that the study will involve 338 participants. Adolescents with ages between 12 and 17 years old, from several middle schools and high schools, will be invited to participate. Assessments will be conducted at four time points: baseline (TThis is the first randomized controlled trial that aims to investigate the efficacy and mechanisms of change of a video-based transdiagnostic REBT universal prevention program, delivered in a school context. The present study has important implications for developing efficient prevention programs, interactive, that will aim to target within the same protocol both anxiety and depressive symptoms.NCT02756507. Registered on 25 April 2016.ClinicalTrials.gov:  Anxiety and depressive disorders are highly prevalent in adolescents . These dPreventing internalizing problems in adolescents could be an important aspect to target, as there is evidence that often anxiety and depressive disorders remain undetected, or even when they are diagnosed, a small percentage of adolescents receive adequate treatment , 11. SevA main limitation of the existent universal prevention programs is the fact that there are no cross-over effects, specifically only those outcomes that are targeted  improve, with no significant changes in the non-targeted condition . These fRational emotive behavior therapy (REBT) , 22, a fGiven the existent barriers in the access to mental health services, communication technologies  can overcome such barriers, improving the availability of mental health services in remote areas and also, reduce the costs . DeliverGiven recent developments in communication technologies, mental health prevention programs for adolescents could overcome existent barriers in terms of access to evidence-based programs. Universal and indicated prevention programs facilitated through technology  are promising in reducing anxiety, depression, and stress in students from higher education , with inThe present study aims to investigate the efficacy of a video-based transdiagnostic REBT universal prevention program for internalizing problems in Romanian adolescents, implemented in a school setting. Our second aim is to investigate subsequent mechanisms of change, namely, we want to investigate whether maladaptive cognitions are significant mediators of the prevention program’s efficacy.The study design is reported according to the CONSORT 2010  Statement  and the www.randomizer.org) in one of the two treatment conditions: (1) video-based transdiagnostic REBT universal prevention program or (2) waitlist control. Assessments will be conducted at four time points: baseline (T1), post-intervention (T2), 3 months follow-up (T3) and 12 months follow-up (T4). Figure A two-arm cluster randomized controlled trial (RCT) will be conducted. Participants’ school classes will be randomized by a computer software ;Not Romanian-speaking in order to understand the content of the prevention program and to complete the assessments.www.randomizer.org), with an equal number of experimental and control groups within one school. Children will be assigned to either video-based transdiagnostic REBT universal prevention or to waitlist control condition. Allocation concealment will be done by an independent researcher, using opaque, sealed envelops that will be opened sequentially. Blinding of participants, researchers and assessors will be maintained.Randomization will be applied within classes, using a random numbers generator (d = .35) according to the results or previous research, for a two-tails between-group comparison at posttest, with an alpha of .05 and a power of 80%. As we expected to have a 30% dropout rate, we will recruit 169 participants in each group.A priori power analyses using G*Power version 3.1.5  indicateParticipants in the waiting list will not receive an intervention. They will complete assessments at all four time points. They could receive the intervention after the experimental group has completed all the assessments.The authors, both being certified in REBT from Albert Ellis Institute, New-York, have developed a transdiagnostic REBT protocol for this study by adjusting existent protocols for internalizing problems in adolescents , 44. NamPsychoeducation regarding internalizing problems. In this session, adolescents will be provided with information regarding internalizing problems. Videos regarding different internalizing symptoms will be presented and discussions regarding different types of symptoms will be initiated with the adolescents. Also, they will be informed about the program and about their involvement in this program.Session 1. Anxious and depressive symptoms identification and relaxation exercises. In this session, adolescents will learn how to recognize anxiety and depression. Also, they will learn the four levels on which such emotions appear: physiological, subjective, cognitive, and behavioral. They will learn the importance of relaxation exercises in order to decrease physiological arousal and they will practice in class a relaxation exercise.Session 2. The ABC model of distress. In this session, adolescents will be thought the ABC model of distress, where A stands for activating events, B stands for beliefs and C stands for consequences. They will learn to distinguish between rational and irrational beliefs, functional and dysfunctional emotions, and discover how different irrational beliefs  are related to different negative dysfunctional emotions  according to the REBT theory. Finally, they will practice examples in which different activating situations are related to different emotions through the influence of irrational beliefs.Session 3. Cognitive restructuring. In this session, adolescents will identify, learn how to dispute their irrational beliefs logically , empirically  and pragmatically . Finally, participants will learn how to dispute their irrational beliefs  and to replace them with rational beliefs  associated with functional emotions and adaptive behaviors. Namely, in this session, the ABC model is integrated with other two important components, namely Disputation (D) and Effective new responses (E) , thus turning into the extended ABCDE model.Session 4. Exposure, behavioral activation and problem-solving. In this session, adolescents will learn how avoidance perpetuates anxiety, and the difference between exposure and avoidance. They will build an exposure hierarchy comprised of fear ladders, with a gradual increase of the distress after disputing their irrational beliefs . The will learn the role of behavioral activation in decreasing depressive mood and will be encouraged to do enjoyable things in order to decrease their depressive symptoms. The second part of this session, adolescents will learn the difference between emotional and practical problems. In this sense, they will learn how to solve practical problems by applying a problem-solving protocol.Session 5. Maintenance of therapeutic gains and relapse prevention. Adolescents will learn how to set realistic expectations regarding the occurrence of internalizing symptoms, and instead of thinking that they will never feel anxiety or depression they will learn that negative emotions can occur, however by identifying and restructuring their irrational beliefs, they will be able to turn those negative dysfunctional emotions into negative functional emotions . Finally, the last session will be a booster session, in which a summary of the program will be presented in order to review all the strategies that adolescents acquired throughout the prevention program so as to prepare them to handle future challenges.Session 6. Homework assignments will be given after each session and participants will have to monitor their emotions , their thinking patterns , dispute their irrational beliefs and replace them with rational beliefs , practice relaxation exercises  at home and in other settings, practice exposure in imaginary and in vivo , practice behavioral activation.N = 5) will be Masters Students, psychologists trained in CBT, under supervision. Therapists will be trained before conducting the sessions with the adolescents and weekly supervision sessions will be conducted with the team.Therapists (1 (baseline), T2 (post-intervention), T3 (3 months follow-up) and T4 (12 months follow-up). An overview of the instruments that will be used for baseline, post-treatment, 3 months follow-up and 12 months follow-up is provided in Fig. Adolescents will complete all the assessments at school, in their classrooms, in the presence of a research assistant, blind to group allocation, that will provide assistance if needed. All the instruments will be applied in a paper and pencil format. Adolescents will complete assessments at four time-points: TBoth adolescents and their parents will complete a questionnaire regarding their demographic characteristics, such as gender, age, urban or rural residency and completed years of education.Never true and 3 means Often true. This scale has four subscales, namely: Physical Symptoms, Social Anxiety, Harm Avoidance and Separation Anxiety/Panic.The Multidimensional Anxiety Scale for Children (MASC)  will be Beck Youth Inventories™ -Second Edition for Children and Adolescents (BYI-II) [Never and 3 = Always. The total score is obtained by summing the 20 items.The depression subscale of the (BYI-II)  will be Not true and 2 means Certainly true. This instrument has five scales: Emotional problems, Conduct problems, Hyperactivity, Peer problems, and Prosocial behavior. This scale allows to compute a Total difficulties score, by summing the first three previously mentioned scales; also, two total scores for Internalizing  and Externalizing  can be computed [The Strengths and Difficulties Questionnaire (SDQ)  self-repcomputed . We willNot at all and 4 means All the time. Total negative automatic thoughts score is computed by summing the three subscales, namely: Physical Threat, Social Threat, and Personal Failure, consisting of 40 items, while total positive automatic thoughts scale consists of 10 items. This instrument showed adequate psychometric properties  previously [The Children’s Automatic Thoughts Scale – Negative/Positive (CATS-N/P)  will be eviously .The Questionnaire for Measuring Health-Related Quality of Life in Children and Adolescents, The Revised Version – adolescent version (Kiddo-KINDL)  will be Not at all, 9 = Very much) while for two items participants will have to choose a percentage between 0% and 100% for expected improvement in their problems.We translated and adapted The Credibility/ Expectancy Questionnaire  in orderDon’t agree at all and 5 = Very much agree). Higher scores on the Satisfaction with treatment scale will indicate greater levels of satisfaction with the program.The Romanian version of the Client Satisfaction Scale, that has been previously used to assess satisfaction with an Internet-delivered CBT , will bed , three months follow-up (T3) and twelve months follow-up (T4), continuous analysis will be conducted using intent-to-treat (ITT) principle the last observation carried forward. In order to assess the efficacy of the video-based transdiagnostic REBT universal prevention program to reduce anxiety and depressive outcomes as compared with the control group over time, we will use mixed effects models . For effect size data we will use Cohen’s d = .80; ). Mediatd = .80; .All the group sessions will be conducted in schools by five registered psychologists, Masters Students. Weekly supervision sessions will be conducted with the therapists in order to solve potential issues that could result in the administration of the intervention. Treatment fidelity and therapists’ adherence to the protocol will be assessed by randomly listening to audio recordings of the sessions.This trial will be conducted in compliance with the Declaration of Helsinki . The stuThis trial’s results will be submitted for publication in peer-reviewed journals, focusing on primary and secondary outcomes results, as well as on mechanisms of change. Also, results will be presented at national and international conferences.Internalizing problems, namely anxiety and depressive disorders are prevalent conditions in adolescents , associaThere are several limitations that we should consider. First, as we rely only on self-reported data, potential bias can result in the case of adolescents. Another important limitation could be the lack of a placebo condition. However, as this will be the first trial comparing the efficacy of a transdiagnostic video-delivered REBT prevention program, we aim to compare it with a waitlist condition and intend to extend this study and compare it with an active placebo or a traditional face-to-face program. Another limitation is related to the possible dropout at 12-month follow-up assessment, as it is possible that several adolescents entering high school will change their schools or live in a different city.In conclusion, this is the first study that aims to investigate the efficacy of a video-based transdiagnostic REBT universal prevention program in reducing internalizing problems in adolescents. Also, it is the first study that integrated REBT with technology in the format of a video-based program aimed to reduce anxiety and depression outcomes in youths. Video-based interventions take into consideration young peoples’ predilection to use technology.Moreover, as indicated in a recent review study analyzing school mental health programs delivered for more than 27 million of children , most ofParticipants’ recruitment is still ongoing.


Pseudomonas aeruginosa. Here we demonstrate that promysalin is active against Gram-positive and Gram-negative bacteria using a microdilution assay. Promysalin acts on Gram-positive bacteria with a mechanism of action involving cell membrane damage with leakage of intracellular components. The evaluation of MICs and MBCs on 11 promysalin analogs, synthesized utilizing diverted total synthesis, allowed the identification of the structural moieties potentially involved in cell membrane interaction and damage. The mechanism of action of promysalin against Gram-negative bacteria is still not clarified, even if a synergistic effect with the bisguanidine chlorhexidine on cell membrane disruption has been observed.Promysalin was previously described as a narrow spectrum molecule with a unique species-specific activity against  Antibiotics do not have species-specific activity, but are characterized by a wide spectrum of activity against several bacterial species and therefore they severely impact on microbiota composition2. It is well known that perturbations to the microbiota, such as those caused by the use of oral antibiotics, deplete the commensal microbiota, allowing pathogens to proliferate, thus determining gastrointestinal inflammations3. In this context, promysalin, a salicylate-containing Pseudomonas putida antibiotic, was described to selectively target members of the genus Pseudomonas, without affecting the viability of other Gram-negative and Gram-positive bacteria5. More recently6, diverted total synthesis of promisalyn analogs demonstrates that the bioactivity of the molecule is sensitive to changes within its hydrogen bond network. Furthermore, it was reported that promysalin inhibits the production of the siderophore pyoverdine which is often linked to virulence. Therefore, the inhibition of pyoverdine by promysalin was considered an interesting point in order to develop novel antivirulence therapy6.The recent studies that highlight the long term effects on human physiology and health of the early exposure to antibioticsPseudomonas4, was described by the authors as a novelty. To the best of our knowledge, there are no other molecules of microbial origin known to be active in a genus-specific way with the exception of a genus-specific molecule which was obtained modifying the original amino acid sequence of the antimicrobial peptide protegrin I through cycles of peptidomimetic synthesis7.The amphiphilic nature of promysalin, prompted us to assume a mechanism of action based on cell membrane interaction. However, the interaction of promysalin with the membrane phospholipid bilayer was never investigated. The narrow spectrum of activity of promysalin against only bacteria of the genus 8, against several Gram-negative and Gram-positive reference species using a validated standard microdilution method9. Additionally, a further aim was to get insights into the mechanism of action of promysalin.The basic purpose of the work outlined here was to investigate the activity of promysalin and analogues, prepared by a recently developed synthesis protocolPseudomonas aeruginosa ATCC 10145 and ATCC 27853, Staphylococcus aureus ATCC 29213 and Streptococcus thermophilus DSM 20617 T, to 256 µg/ml for all Lactobacillus species analyzed. MICs of 64 µg/ml were measured for Enterococcus durans NCDO 956, E. faecalis ATCC 29212, ATCC 19433, and LMG 19456, E. faecium ATCC 19434, Staphylococcus eidermidis ATCC 14990 T and Streptococcus pyogenes ATCC 12344 T. Higher values were determined for the Minimal Bactericidal Concentrations (MBCs) that ranged from 32–64 µg/ml for Bacillus subtilis DSM 347, S. thermophilus DSM 20617 T and S. pneumoniae, to values of 256 or >256 µg/ml for all the other bacterial species analyzed. The overall data indicated that promysalin is sensibly more active on streptococci and lactococci than on lactobacilli. The activity of promysalin detected against Gram-positive microrganisms was in contrast to previous observation4 describing promysalin as an antibiotic which selectively targeted members of the genus Pseudomonas. The reason of these conflicting data is due to the methods used by Li and colleagues4 for the screening of promysalin sensitive bacteria, the agar diffusion test. In the SI  in the same way as when they were exposed to the biocide chlorhexidine (100 µg/ml)  category, is a cationic surfactant whose mechanism of action implies the destruction of the lipid bilayer in the bacterial cell membrane11. Benzalkonium chloride, likewise chlorhexidine determined the same loss of membrane integrity in S. thermophilus and in the Gram-negative Pseudomonas aeruginosa ATCC 10145  showed an increase of membrane damage compared to that obtained exposing cells to high chlorhexidine concentration  compared to P. aeruginosa . We therefore conclude that promysalin and surfactin might have the same mechanisms of action on cell membrane of Gram-positive bacteria.The amphiphilic nature of promysalin may allow an effect on bio-membranes similar to those exerted by microbial lipopetides such as surfactinSaccharomyces cerevisiae BC1 were exposed to promysalin -carboxyfuorescein succinimidyl ester (cFSE) using the cFDASE as a precursor molecule16. The membrane-permeating cFDASE is cleaved by intracellular esterases and the resultant cFSE molecules are conjugated to the aliphatic amines of intracellular proteins17. cFSE labelled S. thermophilus cells were exposed to promysalin or chlorhexidine and the cell membrane damage together with the leakage of cFSE fluorescence outside the cells was monitored by flow cytometry and by a standard fluorometer. As shown in Fig. With the aim to verify if membrane damage was followed by the release of intracellular material, 18. Noteworthy, gramicidin was not effective in creating a membrane damage or a cFSE-fluorescence leakage in S. thermophilus DSM 20617 T . As we noticed that promysalin was quite unstable under acidic conditions8, we also synthesized the cyclized analogue, containing a more rigid tetrahydro-9-oxa-aza-cyclopentanaphthalen-4-one ring (compound 8), by treatment with TFA. All analogs are shown in Fig. The hypothesis on promysalin mechanisms of action formulated above, prompted us to gain insights into the most relevant structural features affecting the damage of the cellular phospholipid bilayer. We recently reported a total synthesis of promysalinPseudomonas aeruginosa ATCC 10145, Staphylococcus aureus ATCC 29213, and S. thermophilus DSM 20617 T. MICs and MBCs values are reported in Table 2, 3, 4, 7 and 8. MBC values below 256 µg/ml were measured only for S. thermophilus strain and for analogs 2, 3, 4, 7 and 8, thus suggesting that for the other species promysalin and the analogs had a bacteriostatic rather than a bactericidal effect. Due to the high MIC values measured for analogs 9–11, we conclude that the salicylate fragment and the dehydroproline moiety are essential for the antibacterial activity of promysalin. It is also interesting to note that analog 8, in contrast to previous observation of Steele and colleagues6, retained a significant activity against Pseudomonas aeruginosa ATCC 10145, Staphylococcus aureus ATCC 29213, and S. thermophilus DSM 20617 T. Analog 5 and 6 showed a significant activity reduction especially when tested against the Gram-positive Staphylococcus aureus ATCC 29213, and S. thermophilus DSM 20617 T  and the methyl ether (4) retained a remarkable membrane damage activity and a remarkable reduction of viability   retained an interesting reduction of viability (54%), but it showed a strong reduction of the ability to interfere with the membrane probably as a consequence of the acquired structural rigidity. The fully functionalized myristamide chain was confirmed mandatory to maintain the activity, and the lipophilic ester derivatives  determined a remarkable reduction of membrane damage confirmed by a weak reduction of cell viability . The C-2 dehydroxylation (3) was found to be detrimental, resulting in a negligible cell viability reduction despite the moderate cell membrane damage observed. Nevertheless, the exposure of S. thermophilus to compound 3 showed the highest % of cells with slightly damaged cell membrane  retained most of the membrane damage activity and cell viability reduction. To summarize, each portion of the parent molecule seemed to play an important role, only the modification of salicylate fragment being tolerated.To evaluate the role on cell membrane damage of the structural modifications, we tested the analogs that retained MIC values equal or lower than 128 µg/ml in Pseudomonas by agar diffusion assay4, showed a wide spectrum of activity when tested by a standard microdilution method for drug susceptibility testing9. Using the microdilution method, promysalin showed to be active against Gram-negative and Gram-positive bacteria with comparable MIC values. Exposing cell suspensions to promysalin, we were able to detect by PI staining a damage of the cell membrane which was clearly evident in all Gram-positive strains tested, and some Gram-negative strains. However, a moderate or no membrane damage was detectable respectively in P. stutzeri LMG 2333 and in P. aeruginosa ATCC 10145, used as representative strains of Gram-negative bacteria, even if they were both sensitive to promysalin4  prior to be used in biological assays.Promysalin synthesis was carried out as described previouslyBacteria strains and yeast were maintained at −80 °C and cultivated in the appropriate media as reported in the Supplementary Information.8. The detailed procedure is described in the Supplementary Information.Minimum inhibitory concentration (MIC) was determined using the standard microdilution method for drug susceptibility testing2HPO4 1,44 g/L; KH2PO4 0.24 g/L; pH 7.4) to a final concentration of 108 events per ml. The cell suspension was diluted to 106 events/ml and then exposed to promysalin (100 µg/ml) or its derivative analogues (100 µg/ml), chlorhexidine (100 µg/ml) (Sigma-Aldrich) or benzalkonium chloride (100 µg/ml) (Sigma-Aldrich) at 37 °C. The cell suspension was also exposed to a DMSO control. At the time requested, a sample was collected and subjected to SYBR Green I/PI double staining and analysis by flow cytometry and, when necessary, to a standard plate count in the appropriate medium. The detailed flow cytometry protocol is reported in the Supplementary Information. To evaluate whether membrane damage was linked to cell leakage of intracellular components, microbial cells grown for 18 h in the appropriate medium in Petri dishes were collected and diluted in PBS to a final concentration of 108 per ml. The obtained cell suspension was supplemented with 4 µM cFDASE , which is a precursor molecule of cFSE. The cFSE fluorescence intensity of stained cells was recovered by flow cytometry in the FL1 channel . The cFSE-labeled cell suspension was then exposed to promysalin (100 µg/ml) or its derivative analogues (100 µg/ml), chlorhexidine (100 µg/ml) (Sigma-Aldrich) or benzalkonium chloride (100 µg/ml) (Sigma-Aldrich) at 37 °C. As a control, the cFSE-labeled cell suspension was also exposed to a volume of DMSO solvent equal to that used for promysalin and its derivative analogues. At the time point, two samples of each cell suspension were collected: i) one sample was labeled with PI as described above, incubated at room temperature for 15 min, and analyzed by flow cytometry. The second sample was used to measure cFSE-fluorescence cell leakage. In this case the sample was centrifuged , and the cell-free supernatant transferred to a 96-microtiter plate for measurement of cFSE-fluorescence in a Victor 3 fluorometer (PerkinElmer). The fluorescence data were calculated as the average of three independent assays and expressed in arbitrary units of fluorescence ± the standard deviation. Further details are described in the Supporting Information.To evaluate whether membrane damage was linked to cell leakage of intracellular components, microbial cells grown for 18 h in the appropriate medium in Petri dishes were collected and diluted in sterile filtered (0.2 µm) phosphate-buffered saline (PBS) (NaCl 8 g/L; KCl 0.2 g/L; NaSupplementary  Information


In many resource-limited settings, emergency medicine (EM) is underdeveloped and formal EM training limited. Residencies and fellowships are an ideal long-term solution but cannot meet immediate needs for emergency providers, while short-term programs are often too limited in content. We describe a third method successfully implemented in Haiti: a medium-duration certificate program to meet the immediate need for emergency specialists.In conjunction with the Haitian Ministry of Health and National Medical School, we developed and implemented a novel, 6-month EM certificate program to build human resources for health and emergency care capacity. The program consisted of didactic and supervised clinical components, covering core content in EM. Didactics included lectures, simulations, hands-on skill-sessions, and journal clubs. Supervised clinical time reinforced concepts and taught an EM approach to patient care.Fourteen physicians from around Haiti successfully completed the program; all improved from their pre-test to post-test. At the end of the program and 9-month post-program evaluations, participants rated the program highly, and most felt they used their new knowledge daily. Participants found clinical supervision and simulation particularly useful.Key components to our program’s success included collaboration with the Ministry of Health and National Medical School, supervised clinical time, and the continual presence of a course director. The program could be improved by a more flexible curriculum and by grouping participants by baseline knowledge levels.Medium-duration certificate programs offer a viable option for addressing immediate human resource gaps in emergency care, and our program offers a model for implementation in resource-limited settings. Similar options should be considered for other emerging specialties in resource-limited settings. There is a tremendous need for improved emergency services in low- and middle-income countries (LMICs) –3 to redThough the WHO has endorsed emergency care as “an essential part of integrated healthcare delivery,”  emergencNo single best practice has been established to meet the immediate EM training needs in LMICs. Directly exporting training programs from high-resource settings is not a viable solution, given differences in human resources and health system development. Recently, several LMICs have opened EM residencies and fellowships, ranging in duration from 1 to 6 years –13. OverAn alternate training approach is short courses of several days to weeks , 15, oftWe propose a novel third method: a medium-duration certificate to meet the immediate need for emergency specialists. We describe the implementation of such a certificate program in Haiti, a low-income country with representative human resource and health system gaps in emergency care.There are five medical schools in Haiti, who together graduate approximately 350 students annually. Each has a 6 or 7-year curriculum with the final year devoted entirely to clinical rotations. In addition, the health system welcomes Haitians who have graduated from medical schools abroad, including from Cuba through the Haitian-Cuban cooperation. There is no formal EM training at either the medical or nursing schools throughout the country.To become licensed, medical school graduates complete a mandatory social service year, in which they are assigned to work at hospitals around the country by the ministry of health. Though some placements are urban with more senior physicians, many placements are rural and new graduates may be the only physician at the facility. New graduates are not formally supervised during this social service year; after completion of social service, they become licensed as generalist physicians.Licensed generalist physicians can apply for a residency program, but less than a third of the applicants get a residency position . Although Haiti has increased its graduate medical education capacity since the 2010 earthquake, residency opportunities remain limited while the number of applicants increases. Physicians who do not apply for or receive a residency acceptance continue to work as generalist physicians. Generalists work in both the public and private sector at primary and secondary levels of health care.Haiti has few post-graduate medical training programs and, prior to October 2014, had no EM residency program or emergency physicians . EM servIn response to requests from and in collaboration with the Ministry of Health and National Medical School, we created a 6-month Certificate in EM to train physicians in the core competencies of the specialty and address a national training gap.Fourteen physicians participated in the certificate program, including all eight HUM ED physicians and six physicians with leadership roles in other Haitian EDs . All three other academic hospitals in Haiti sent participants, as did two nearby district hospitals and a non-governmental organization trauma hospital in Port-au-Prince. Hospitals nominated their representative individual participants, chosen for their leadership skills and likelihood to teach others. Clinical supervision needs dictated the maximum number of participants. Four participants had prior residency training , and the remainder were generalist physicians. None of the participants had prior emergency medicine training. Though all participants were currently working in EDs, some  had worked in EDs for less than 1 year. The certificate program was free of charge to all participants, who remained on full salary at their home institutions during the program.The program consisted of didactic lectures, simulation, and clinical supervision. The didactic component was held every other week, allowing participants to work full-time at their home hospitals and reduce transportation costs. To graduate, participants were required to attend 75% of didactic lectures, complete 180 supervised clinical hours, take a pre- and post-test, prepare and give one lecture, and submit a case and procedure log. The pre- and post-tests were written by a panel of emergency physicians (EPs) with global health expertise and experience in Haiti. Questions were peer-reviewed by the course directors.The 96-h didactic program focused on core EM competencies and included lectures , international EPs provided clinical supervision: two US-trained EPs together provided full-time program leadership and supervision, and eight visiting faculty each volunteered for 3–4 weeks. On average, two supervising emergency physician (EP) faculty were at HUM at any time throughout the program. Participants completed at least 180 h of supervised clinical time at HUM. Supervision schedules were designed to minimize participant travel, assure oversight, and avoid ED overstaffing.Participants maintained a case log, documenting both 100 supervised cases and pre-specified numbers of essential procedures, such as adult and pediatric resuscitations, thoracentesis, and splinting/casting Table . As an eTo minimize costs, we relied on volunteer visiting faculty for whom we subsidized only in-country expenses, although this resulted in frequent turnover of clinical supervisors. The co-course directors were also the department co-chairs and were not hired specifically for this course. Participants were not compensated; participants not from HUM received local food and housing but not transport. Clinical time in the HUM ED did not add additional operating costs.All 14 participants completed the certificate program. One year later, 11 were still working in EDs. All participants improved on their 56 question pre-/post-test with an average improvement of 15 percentage points , cardiovascular emergencies and ECG interpretation, pediatric emergencies, dyspnea, and the philosophy of emergency medicine .Participants were asked to anticipate barriers to applying their new knowledge. One common barrier was resource limitations at their home institutions compared to HUM. Others included patient education, late-stage presentations, motivating other staff, and the perceived difficulty in changing culture at their home institutions.Nine months after the program, a second evaluation was sent to the participants. Nine (64%) responded, all of whom still worked in EDs. Most reported using knowledge from the certificate program on a daily basis . When asked to rate the importance of their supervised clinical time on their education, eight (89%) felt it was “very important.” Program components valued at 9 months included the supervised clinical experience, simulations and structured debriefs, and specific clinical topics including orthopedic emergencies, shock, trauma management, and ECG interpretation.This medium duration, 6-month certificate program in Haiti, represents a novel approach to address gaps in human resources for health and can be replicated for emergency care and other new specialties in similar settings. Notably, the program improved participants’ EM knowledge and was well received by participants, the Haitian government, and the national medical school.Several structural components were critical to the program’s success. First, the engagement of the Haitian Ministry of Health and National Medical School allowed official certification of the program and facilitated participation of physicians from multiple public hospitals. Second, training physicians from throughout the country disseminated knowledge, enhanced recognition of EM as a specialty, and fostered an EM professional network.Third, we recruited both international EPs and national non-EP specialists as program faculty. Visiting EPs were crucial for teaching core EM content and role-modeling the clinical approach to the emergency patient. For select topics, national specialists from other disciplines taught didactic content related to their expertise, with explicit guidance on learning objectives important for EPs. For example, a Haitian obstetrician-gynecologist lectured on first trimester obstetric emergencies. This involvement in the program increased recognition of EM among other specialties, and their lectures were inherently adapted to local context.Fourth, the continuous presence and leadership of a course director was essential, facilitating coordination and real-time management of contingencies, as well as orienting international EPs to the local standards of care, knowledge level of participants, and successful teaching strategies. This allowed successive visiting faculty to maintain consistent focuses on the strengths and weaknesses of individual participants and difficult content areas. Course directors provided consistency for participants, ensuring that requirements were met and adapting the curriculum as needed.Finally, clinical supervision and simulation were essential to the program’s success. Direct clinical supervision helped participants learn the approach to the emergency patient and apply knowledge from didactics. Topics covered briefly during didactics, such as image interpretation, were explored in depth at the bedside. Importantly, case logs facilitated clinical supervision, gave objective performance measures, and structured the relationship between participants and visiting faculty. In addition, simulation taught simultaneous stabilization, diagnosis, and treatment with a focus on low-frequency, but high-risk emergencies. Simulation also offered a safe environment for students to learn without posing risk to patients.The program could be improved by structuring more flexibility into the curriculum to accommodate events from illnesses to unforeseen opportunities for specialist lectures. Further, some didactic areas  required more time than anticipated. Similarly, due to clinical variability during ED shifts, not all participants met the pre-specified procedure targets, and we offered flexibility in this requirement.Future programs could group participants by baseline knowledge level; the varied baseline knowledge level among our participants necessitated adapting lectures to ensure content was appropriate for all learners. Lastly, translation provided an unexpected challenge, as the hospital English-French translators lacked sufficient medical knowledge to interpret nuanced clinical concepts. In the end, our participants provided real-time translation themselves (by those fluent in English) to maximize group education.Regarding sustainability, our conclusion at the end of the program was that the 6-month certificate alone is not sufficient as a train-the-trainer model. While students improved their clinical EM skills—the primary objective of the course—they did not reach a level sufficient to become teachers themselves. Future iterations should consider alternate ways to develop local trainers, either by repeating the course multiple times, providing additional clinical supervision and accompaniment before or after the course, and/or pairing the certificate program with residency training, so that local residency-trained EPs could ultimately run the certificate course.Medium-duration certificate programs offer a novel approach to address the need for qualified emergency providers in LMICs by offering greater breadth and depth than traditional short courses while expeditiously training more providers than a residency: in the same 13 years needed to graduate 50 residency-trained EPs, an annual certificate program could train 180 providers. In most of Haiti, like many LMICs, generalist physicians with limited training provide front-line emergency care. Even though Haiti now has an EM residency program, placing specialists in remote hospitals will not be realistic anytime in the foreseeable future. Certificate-trained generalist providers could help bridge these human resource gaps. Further, this program could help link remote generalist providers to teaching hospitals where the training is run to create long-term educational support and transfer networks. For these reasons, the Haitian Ministry of Health and National Medical School have requested that the emergency certificate course be offered on an ongoing basis.Some questions remain unanswered. Our program was designed to meet Haiti’s needs. We anticipate the content is generalizable to other LMICs, but this warrants further evaluation, as does knowledge retention after the course. Additionally, the role of a certificate program in a country with a new EM residency is undetermined. The coexistence of certificate-trained and residency-trained physicians could impact the new specialty as it seeks professionalization. Finally, this program was successful for generalist and specialist physicians with no prior EM training, over half of whom had worked in EDs for less than a year. Thus, we anticipate that this program is relevant to all physicians, particularly recent graduates and those with limited prior EM experience. The adaptability of this curriculum to other cadres of providers, such as nurses or midlevel providers, remains to be seen. However, the program’s design, covering core EM content, may allow easy modification for medical students, residents in other specialties, and/or midlevel providers to facilitate task-shifting.The implementation experience and lessons learned from this 6-month curriculum in Haiti offer a starting point for future programs. Given the urgent need for qualified emergency providers in LMICs, medium-duration EM certificate programs can play a critical role in addressing the immediate human resources gap to provide high-quality emergency care. Similar human resource gaps for other medical specialties also exist in LMICs, and medium-term certificate trainings warrant exploration in these fields as well.