Digital Microscopy Augmented by Artificial Intelligence to Interpret Bone Marrow Samples for Hematological Diseases.

Analysis of bone marrow aspirates (BMAs) is an essential step in the diagnosis of hematological disorders. This analysis is usually performed based on a visual examination of samples under a conventional optical microscope, which involves a labor-intensive process, limited by clinical experience and subject to high observer variability. In this work, we present a comprehensive digital microscopy system that enables BMA analysis for cell type counting and differentiation in an efficient and objective manner. This system not only provides an accessible and simple method to digitize, store, and analyze BMA samples remotely but is also supported by an Artificial Intelligence (AI) pipeline that accelerates the differential cell counting process and reduces interobserver variability. It has been designed to integrate AI algorithms with the daily clinical routine and can be used in any regular hospital workflow.

Unfolding and Aggregation Pathways of Variable Domains from Immunoglobulin Light Chains.

Light chain amyloidosis is the most common form of systemic amyloidosis. This disease is caused by the formation and deposition of amyloid fibers made from immunoglobulin light chains. Environmental conditions such as pH and temperature can affect protein structure and induce the development of these fibers. Several studies have shed light on the native state, stability, dynamics, and final amyloid state of these proteins; however, the initiation process and the fibril formation pathway remain poorly understood structurally and kinetically. To study this, we analyzed the unfolding and aggregation process of the 6aJL2 protein under acidic conditions, with temperature changes, and upon mutation, using biophysical and computational techniques. Our results suggest that the differences in amyloidogenicity displayed by 6aJL2 under these conditions are caused by traversing different aggregation pathways, including unfolded intermediates and the formation of oligomers.

Dimers of D76N-ß2-microglobulin display potent antiamyloid aggregation activity.

Self-association of WT ß2-microglobulin (WT-ß2m) into amyloid fibrils is associated with the disorder dialysis related amyloidosis. In the familial variant D76N-ß2m, the single amino acid substitution enhances the aggregation propensity of the protein dramatically and gives rise to a disorder that is independent of renal dysfunction. Numerous biophysical and structural studies on WT- and D76N-ß2m have been performed in order to better understand the structure and dynamics of the native proteins and their different potentials to aggregate into amyloid. However, the structural properties of transient D76N-ß2m oligomers and their role(s) in assembly remained uncharted. Here, we have utilized NMR methods, combined with photo-induced crosslinking, to detect, trap, and structurally characterize transient dimers of D76N-ß2m. We show that the crosslinked D76N-ß2m dimers have different structures from those previously characterized for the on-pathway dimers of ΔN6-ß2m and are unable to assemble into amyloid. Instead, the crosslinked D76N-ß2m dimers are potent inhibitors of amyloid formation, preventing primary nucleation and elongation/secondary nucleation when added in substoichiometric amounts with D76N-ß2m monomers. The results highlight the specificity of early protein-protein interactions in amyloid formation and show how mapping these interfaces can inform new strategies to inhibit amyloid assembly.

Influence of surfactant on glass transition temperature of poly(lactic-co-glycolic acid) nanoparticles.

Poly(D,L-lactic-co-glycolic acid) (PLGA) is one of the most commonly used drug carriers in nanomedicines because of its biodegradability, biocompatibility and low toxicity. However, the physico-chemical characterization and study of drug release are often lacking the investigation of the glass transition temperature (Tg), which is an excellent indicator of drug release behavior. In addition, the residual surfactant used during the synthesis of nanoparticles will change the glass transition temperature. We thus prepared PLGA nanoparticles with polymeric (poly(vinyl alcohol) (PVA)) and ionic (didodecyldimethylammonium bromide (DMAB)) surfactant to investigate their influence on the glass transition temperature. Determination of Tg in dry and wet conditions were carried out. The use of concentrated surfactant during synthesis resulted in a larger amount of residual surfactant in the resulting particles. Increasing residual PVA content resulted in an increase in particle Tg for all but the most concentrated PVA concentrations, while increasing residual DMAB content resulted in no significant change in particle Tg. With the presence of residual surfactant, the Tg of particle and bulk samples measured in wet conditions is much lower than that in dry conditions, except for bulk PLGA containing the ionic surfactant, which may be related to the plasticizing effect of the DMAB molecules. Notably, the Tg of both particles in wet conditions is approaching physiological temperatures where subtle changes in Tg could have dramatic effects on drug release properties. In conclusion, the selection of surfactant and the remaining amount of surfactant are crucial parameters to utilize in designing the physico-chemical properties of PLGA particles.

Impact of Drying Process on the Phenolic Profile and Antioxidant Capacity of Raw and Boiled Leaves and Inflorescences of Chenopodium berlandieri ssp. berlandieri.

C. berlandieri ssp. berlandieri (C. berlandieri) is one of the most common members of the group of plants known as quelites, which are dark leafy greens widely consumed in Mexico. This study aimed to evaluate the impact of two drying procedures (oven drying and freeze-drying/lyophilization) on the polyphenolic composition, antioxidant capacity, and proximal chemical analysis of C. berlandieri leaves and inflorescences (raw or boiled). The results indicated that the raw freeze-dried samples had higher amounts (p < 0.05) of total phenolic compounds, total flavonoids, and antioxidant capacity, mainly in the inflorescence. The oven-dried samples showed an increased concentration of polyphenols after boiling, while the lyophilized samples showed a slightly decreased concentration. The drying process was observed to have little impact on the proximal chemical composition. Quantification by UPLC-DAD-ESI-QToF/MS identified up to 23 individual phenolic compounds, with freeze-dried samples showing higher amounts of individual compounds compared with oven-dried. Procyanidin B2 was found exclusively in the inflorescences. The inflorescences have a higher content of phenolic compounds and greater antioxidant capacity than the leaves. Regardless of the drying process, the leaves and inflorescences of C. berlandieri contain an interesting variety of phenolic compounds that may have beneficial effects on health.

Integral Study of Paramecium caudatum Acute and Chronic Toxicity, Sites of Entry and Distribution, Bioconcentration and Body Burdens of Five Metals.

An integral analysis of the acute and chronic toxicity, bioaccumulation, sites of entry, and distribution of four trace metals: copper, iron, lead, and nickel, and the non-trace metal mercury were performed in the ciliate Paramecium caudatum. Mercury was the fastest metal accumulated, and the most toxic. The sensitivity of Paramecium caudatum to the five metals tested (Cu, Fe, Hg, Ni, and Zn) falls in the range of other ciliate species. We observed similarities between the toxicity of the five metals to the ciliate P. caudatum with the rotifer Euchlanis dilatata: (a) Mercury was the most toxic metal in terms of acute and body burdens. (b) Acute values were very similar in both species, Hg as the most toxic and Fe as the less toxic, (c) the vacuole/ingestion chronic tests were more sensitive than growth inhibition chronic tests. These analyses would ideally help generate safer guidelines for protecting aquatic biota.

Management of Persistent SARS-CoV-2 Infection in Patients with Follicular Lymphoma.

INTRODUCTION: There is no consensus on the management of the coronavirus disease (COVID-19) in patients with secondary immunosuppression due to either an underlying hematological disease or to the effects of immunochemotherapy (ICT). Some of them may present persistent infection with multiple relapses of COVID-19, requiring several admissions. This study evaluated the clinical characteristics and outcomes after treatment of 5 patients with follicular lymphoma (FL), previously treated with ICT, who developed several episodes of COVID-19. METHODS: We analyzed the clinical evolution and response to treatment with antiviral agent, steroids, and convalescent plasma in 5 patients with FL and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) persistent infection. Reverse transcriptase polymerase chain reaction tests and peripheral blood immunophenotype were performed for all patients. RESULTS: All patients required hospitalization due to pneumonia with severity criteria and were re-admitted after a median of 22 days (13-42) from the previous discharge. They all showed B-cell depletion by immunophenotyping, and no traces of immunoglobulin antibodies against SARS-CoV-2 were detected in any of the cases. The survival rate was 80%. CONCLUSION: The combination therapy evidenced clinical benefits, demonstrating its capacity to control infection in immunosuppressed FL patients treated with ICT.

Toxicity tests, bioaccumulation and residuality of pyrethroid insecticides commonly used to control conifer bark beetles in Mexico.

Effectiveness of pyrethroid insecticides against pests including bark beetles (Dendroctonus spp.) is well known, but little is known about their (a) residuality and persistence in forests after application to control an outbreak and (b) ability to bioaccumulate in insects, which could cause adverse effects on the trophic chains. The 24 h lethal toxicity, bioaccumulation factors, and lethal body burdens of the pyrethroid insecticides bifenthrin, deltamethrin, and cypermethrin were evaluated in the bark beetle Dendroctonus mexicanus. The residuality and persistence of the insecticide bifenthrin in the forest ecosystem (soil, water, and sediment) was also monitored after its application in a region infested by bark beetles. We determined whether contamination of soil, water, and sediments occurred near the sanitation site. For D. mexicanus, the most toxic insecticide for adults was bifenthrin for topical application (LC50 = 0.94 mg/L) and cypermethrin for bark application (LC50 = 5.04 mg/L). The insecticide that bioaccumulated the most in the insect body was deltamethrin (622.41 µg g-1 dw) and the insecticide that bioaccumulated the least amount was cypermethrin (183.09 µg g-1 dw). However, cypermethrin was the active substance that presented the lowest body burdens, and therefore required a lower dose to cause death of D. mexicanus adults. Our results demonstrate that prolonged presence of bifenthrin in the forest ecosystem could lead to bioaccumulation in soil, sediment, and insects.

Synergistic effect of chloroquine and copper to the euryhaline rotifer Proales similis.

Chloroquine (CQ) has been widely used for many years against malaria and various viral diseases. Its important use and high potential to being persistent make it of particular concern for ecotoxicological studies. Here, we evaluated the toxicity of CQ alone and in combination with copper (Cu) to the euryhaline rotifer Proales similis. All experiments were carried out using chronic toxicity reproductive five-day tests and an application factor (AF) of 0.05, 0.1, 0.3, and 0.5 by multiplying the 24-h LC50 values of CQ (4250 µg/L) and Cu (68 µg/L), which were administered in solution. The rate of population increase (r, d-1) ranged from 0.50 to 52 (controls); 0.20 to 0.40 (CQ); 0.09 to 0.43 (Cu); and -0.03 to 0.30 (CQ-Cu) and showed significant decrease as the concentration of both chemicals in the medium increased. Almost all tested mixtures induced synergistic effects, mainly as the AF increased. We found that the presence of Cu intensifies the vulnerability of organisms to CQ and vice versa. These results stress the potential hazard that these combined chemicals may have on the aquatic systems. This research suggests that P. similis is sensitive to CQ as other standardized zooplankton species and may serve as a potential test species in the risk assessment of emerging pollutants in marine environments.

Inhibition of bacterial growth through LED (light-emitting diode) 465 and 630 nm: in vitro.

Photobiomodulation has been used to inactivate bacterial growth, in different laser or LED protocols. Thus, the aim of this study was to verify the inhibition of Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli, in ATCC strains and bacteria collected from patients with skin burns, after irradiation with LED; 300 µl of saline solution with bacterial suspension was irradiated at a concentration of 0.5-0.63, by the McFarland scale, after five serial dilutions, with evaluation of pre- and post-irradiation pH and temperature control. The cultures were placed in a bacteriological incubator at 37 °C for 24 h for later counting of colony-forming units (CFU). Data were analyzed by Shapiro-Wilk tests and single-factor ANOVA, with Tukey post hoc (p < 0.05). Both wavelengths and energy densities tested showed inhibition of bacterial growth. The comparison of the irradiated groups (ATCC) with the control group showed the following: S. aureus and P. aeruginosa 465 nm (40 J/cm2) and 630 nm (50 J/cm2) and E. coli 465 nm (40 J/cm2) and 630 nm (30 J/cm2). Among the ATCC S. aureus groups, there was a difference for 630 nm (30 J/cm2) and 465 nm (30, 40, 50 J/cm2). The bacteria from the burned patients were S. aureus (30 and 50 J/cm2) and P. aeruginosa (50 J/cm2). We conclude that different bacterial strains were reduced into colony-forming units after LED irradiation.

Literature Review: Robotic-Assisted Harvest of Deep Inferior Epigastric Flap for Breast Reconstruction.

INTRODUCTION: Robotic-assisted surgery is gaining popularity because of reported improvement in aesthetic outcomes while reducing the occurrence of complications compared with conventional surgical methods. Deep inferior epigastric perforator (DIEP) flap harvesting has a long track record as a viable procedure for autologous reconstruction of the breast. In this literature review, we describe the feasibility of using the robotic platform in DIEP flap harvest. METHODS: The Preferred Reporting Items for Systemic Reviews and Meta-Analysis methodology was to guide the literature review. PubMed and Scopus databases were searched from inception to June 6, 2022. The Medical Subject Heading terms and keywords used to conduct this search are as described: "Robotic AND deep inferior epigastric perforator AND Breast reconstruction." RESULTS: Seven publications, detailing a total of 56 robotic-assisted DIEP flap harvest procedures, were selected for review. Four publications used the transabdominal preperitoneal approach, whereas 2 exclusively used a totally extraperitoneal approach, and 1 compared the 2 approaches. The measured outcomes included technical feasibility of flap harvest in cadavers, viable flap harvest in live patients, harvest time and pedicle dissection time, pedicle length, fascial incision length, donor site pain, need for postoperative narcotic, donor site morbidity, and hernia formation. Overall, the reviewed articles demonstrated successful DIEP flap harvesting without the need for conversion to the conventional open procedure. Postoperative complications were minimal. Robotic DIEP flap harvest was shown to be safe and there were no reports of donor-site morbidity in the studies reviewed. The main advantages of the robotic approach include decreased postoperative pain and length of hospital stay, along with improved aesthetic outcomes. The main disadvantages are increased operative time and cost. CONCLUSIONS: Although at its current iteration, the robotic-assisted DIEP flap is feasible, it may not be practical in all settings. Furthermore, the true benefit of the robotic platform is yet to be determined, as more long-term studies are necessary.

ß-Lactam TRPM8 Antagonist RGM8-51 Displays Antinociceptive Activity in Different Animal Models.

Transient receptor potential melastatin subtype 8 (TRPM8) is a cation channel extensively expressed in sensory neurons and implicated in different painful states. However, the effectiveness of TRPM8 modulators for pain relief is still a matter of discussion, since structurally diverse modulators lead to different results, depending on the animal pain model. In this work, we described the antinociceptive activity of a ß-lactam derivative, RGM8-51, showing good TRPM8 antagonist activity, and selectivity against related thermoTRP channels and other pain-mediating receptors. In primary cultures of rat dorsal root ganglion (DRG) neurons, RGM8-51 potently reduced menthol-evoked neuronal firing without affecting the major ion conductances responsible for action potential generation. This compound has in vivo antinociceptive activity in response to cold, in a mouse model of oxaliplatin-induced peripheral neuropathy. In addition, it reduces cold, mechanical and heat hypersensitivity in a rat model of neuropathic pain arising after chronic constriction of the sciatic nerve. Furthermore, RGM8-51 exhibits mechanical hypersensitivity-relieving activity, in a mouse model of NTG-induced hyperesthesia. Taken together, these preclinical results substantiate that this TRPM8 antagonist is a promising pharmacological tool to study TRPM8-related diseases.

Environment affects starch composition and kernel hardness in temperate maize.

BACKGROUND: Protein percentage and kernel weight affect the endosperm hardness of maize grains. However, changes in starch composition could also modify kernel hardness, which is often predicted through milling ratio. The objective of this work was to evaluate the relationship between changes in starch composition and endosperm hardness, and to assess the effects of protein content and kernel weight on that relationship. For this, we used information obtained from three temperate hybrids in multi-environmental experiments, as well as experiments conducted under controlled conditions designed to modify starch composition. Milling ratio was determined as maize kernel hardness predictor in both experiments and it was correlated with grain weight and grain composition. RESULTS: In both experiments, milling ratio presented a significant Spearman correlation coefficient with amylose/starch ratio. Milling ratio was significantly related to the amylose/starch ratio using a simple fit with datasets from different experiments and hybrids. Increases in amylose/starch ratio were associated with increases in milling ratio. CONCLUSION: Starch composition was related to milling ratio variations among hybrids of different aptitude for dry milling through different environments, regardless of protein content. Thus, increases in the amylose/starch ratio were related to increases in the milling ratio. © 2022 Society of Chemical Industry.

The role of the IT-state in D76N ß2-microglobulin amyloid assembly: A crucial intermediate or an innocuous bystander?

The D76N variant of human ß2-microglobulin (ß2m) is the causative agent of a hereditary amyloid disease. Interestingly, D76N-associated amyloidosis has a distinctive pathology compared with aggregation of WT-ß2m, which occurs in dialysis-related amyloidosis. A folding intermediate of WT-ß2m, known as the IT-state, which contains a nonnative trans Pro-32, has been shown to be a key precursor of WT-ß2m aggregation in vitro However, how a single amino acid substitution enhances the rate of aggregation of D76N-ß2m and gives rise to a different amyloid disease remained unclear. Using real-time refolding experiments monitored by CD and NMR, we show that the folding mechanisms of WT- and D76N-ß2m are conserved in that both proteins fold slowly via an IT-state that has similar structural properties. Surprisingly, however, direct measurement of the equilibrium population of IT using NMR showed no evidence for an increased population of the IT-state for D76N-ß2m, ruling out previous models suggesting that this could explain its enhanced aggregation propensity. Producing a kinetically trapped analog of IT by deleting the N-terminal six amino acids increases the aggregation rate of WT-ß2m but slows aggregation of D76N-ß2m, supporting the view that although the folding mechanisms of the two proteins are conserved, their aggregation mechanisms differ. The results exclude the IT-state as the origin of the rapid aggregation of D76N-ß2m, suggesting that other nonnative states must cause its high aggregation rate. The results highlight how a single substitution at a solvent-exposed site can affect the mechanism of aggregation and the resulting disease.

Molecular profiling of advanced solid tumours. The impact of experimental molecular-matched therapies on cancer patient outcomes in early-phase trials: the MAST study.

INTRODUCTION: Molecular-matched therapies have revolutionized cancer treatment. We evaluated the improvement in clinical outcomes of applying an in-house customized Next Generation Sequencing panel in a single institution. METHODS: Patients with advanced solid tumors were molecularly selected to receive a molecular-matched treatment into early phase clinical trials versus best investigators choice, according to the evaluation of a multidisciplinary molecular tumor board. The primary endpoint was progression-free survival (PFS) assessed by the ratio of patients presenting 1.3-fold longer PFS on matched therapy (PFS2) than with prior therapy (PFS1). RESULTS: Of a total of 231 molecularly screened patients, 87 were eligible for analysis. Patients who received matched therapy had a higher median PFS2 (6.47 months; 95% CI, 2.24-14.43) compared to those who received standard therapy (2.76 months; 95% CI, 2.14-3.91, Log-rank p = 0.022). The proportion of patients with a PFS2/PFS1 ratio over 1.3 was significantly higher in the experimental arm (0.33 vs 0.08; p = 0.008). DISCUSSION: We demonstrate the pivotal role of the institutional molecular tumor board in evaluating the results of a customized NGS panel. This process optimizes the selection of available therapies, improving disease control. Prospective randomized trials are needed to confirm this approach and open the door to expanded drug access.

Paradoxical effect of SARS-CoV-2 infection in patients with immune thrombocytopenia.

Thrombocytopenia has been identified as a common complication of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection in the general population. In an attempt to determine the impact of coronavirus disease 2019 (COVID-19) in patients with immune thrombocytopenia (ITP), a retrospective single-centre study was performed. Thrombocytosis was observed in patients with chronic ITP after SARS-CoV-2 infection, frequently needing treatment adjustment or even discontinuation of therapy. Relapses and newly diagnosed cases showed a fast response after initial treatment compared to ITP. Reduced immune activity due to lymphopenia during COVID-19 could explain this paradoxical effect, although further studies are needed.

Total syntheses and antiproliferative activities of prenostodione and its analogues.

A high-yielding total synthesis of the indole alkaloid prenostodione was completed in 4 steps and 44% overall yield from 1H-indole-3-carboxylic acid. The expedient syntheses of prenostodiones containing distinct substituents at the para position of the phenyl frame underscored the scope of this methodology. The cytotoxic activities of the tert-butyl esters of prenostodione analogues were tested using six tumor cell lines. Preliminary structure-activity studies revealed the importance of the identity of the aromatic substituent at the C-4 position for cytotoxic activity. The IC50 values of these compounds were found to compare satisfactorily with those of the commercially available drugs etoposide and cisplatin. Furthermore, the compounds with, respectively, -OMe (14d) and -NO2 (14f) groups at C-4 were more selective than these control compounds in PC-3, K-562, and MCF-7 cells. Also, computational studies were carried out to determine the ADMET profiles and passive membrane permeabilities of the compounds. The results suggested the promise of 14d and 14f as hit compounds for the development of new anticancer agents.

Frontal Sinus Fracture Management Meta-analysis: Endoscopic Versus Open Repair.

INTRODUCTION: Frontal sinus fracture management continues to be a point of controversy. Many systematic reviews have been done, but meta-analyses comparing various approaches to frontal sinus fractures are scarce. Our study focuses on open surgical repair versus endoscopic repair of frontal sinus fractures. MATERIALS AND METHODS: A MEDLINE search was performed to identify prospective RCTs, non-RCTs, guidelines, case-control, and observational studies published in English before April 2019. Search terms included (1) frontal sinus, (2) skull fractures, (3) frontal bone, alone or in combination. An in-depth review was conducted to identify publications relevant to this analysis. Studies that included pediatric patients, case reports, and review articles without original data were excluded. Postoperative outcomes included were cosmesis, sinus function, mucocele, sinusitis, cerebrospinal fluid leak, meningitis, and brain abscess. Meta-analysis of proportions, Fisher exact test, and relative risks were calculated. RESULTS: Seven studies were ultimately included in the meta-analysis. The assessment of heterogeneity indicated that the studies are comparable. The weighted outcome proportions of patients with postoperative cosmetic deformity and mucocele are found to be significantly higher in patients who underwent open repair compared to patients who received endoscopic treatment. No significant difference between the 2 intervention groups in regards to the other outcomes. CONCLUSION: Based on this meta-analysis, no definitive conclusions regarding superiority of one approach over the other can be made. There are currently no universally accepted algorithms that aid in the decision to proceed with either approach. It is likely that these 2 techniques will come to serve different roles in treatment, as they can each be utilized to achieve different goals. At this time, the only recommendation is to proceed with whichever technique can be safely performed based upon surgeon experience and fracture pattern.

Design and Validation of a Scalable, Reconfigurable and Low-Cost Structural Health Monitoring System.

This paper presents the design, development and testing of a low-cost Structural Health Monitoring (SHM) system based on MEMS (Micro Electro-Mechanical Systems) triaxial accelerometers. A new control system composed by a myRIO platform, managed by specific LabVIEW software, has been developed. The LabVIEW software also computes the frequency response functions for the subsequent modal analysis. The proposed SHM system was validated by comparing the data measured by this set-up with a conventional SHM system based on piezoelectric accelerometers. After carrying out some validation tests, a high correlation can be appreciated in the behavior of both systems, being possible to conclude that the proposed system is sufficiently accurate and sensitive for operative purposes, apart from being significantly more affordable than the traditional one.

Phenylalanine-Derived ß-Lactam TRPM8 Modulators. Configuration Effect on the Antagonist Activity.

Transient receptor potential cation channel subfamily M member 8 (TRPM8) is a Ca2+ non-selective ion channel implicated in a variety of pathological conditions, including cancer, inflammatory and neuropathic pain. In previous works we identified a family of chiral, highly hydrophobic ß-lactam derivatives, and began to intuit a possible effect of the stereogenic centers on the antagonist activity. To investigate the influence of configuration on the TRPM8 antagonist properties, here we prepare and characterize four possible diastereoisomeric derivatives of 4-benzyl-1-[(3'-phenyl-2'-dibenzylamino)prop-1'-yl]-4-benzyloxycarbonyl-3-methyl-2-oxoazetidine. In microfluorography assays, all isomers were able to reduce the menthol-induced cell Ca2+ entry to larger or lesser extent. Potency follows the order 3R,4R,2'R > 3S,4S,2'R ≅ 3R,4R,2'S > 3S,4S,2'S, with the most potent diastereoisomer showing a half inhibitory concentration (IC50) in the low nanomolar range, confirmed by Patch-Clamp electrophysiology experiments. All four compounds display high receptor selectivity against other members of the TRP family. Furthermore, in primary cultures of rat dorsal root ganglion (DRG) neurons, the most potent diastereoisomers do not produce any alteration in neuronal excitability, indicating their high specificity for TRPM8 channels. Docking studies positioned these ß-lactams at different subsites by the pore zone, suggesting a different mechanism than the known N-(3-aminopropyl)-2-[(3-methylphenyl)methoxy]-N-(2-thienylmethyl)-benzamide (AMTB) antagonist.