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Expanding the range of Protospacer Adjacent Motifs (PAMs) recognized by CRISPR-Cas9 is essential for broadening genome-editing applications. Here, we combine molecular dynamics simulations with graph-theory and centrality analyses to dissect the principles of PAM recognition in three Cas9 variants - VQR, VRER, and EQR - that target non-canonical PAMs. We show that efficient recognition is not dictated solely by direct contacts between PAM-interacting residues and DNA, but also by a distal network that stabilizes the PAM-binding domain and preserves long-range communication with REC3, a hub that relays signals to the HNH nuclease. A key role emerges for the D1135V/E substitution, which enables stable DNA binding by K1107 and preserves key DNA phosphate locking interactions via S1109, securing stable PAM engagement. In contrast, variants carrying only R-to-Q substitutions at PAM-contacting residues, though predicted to enhance adenine recognition, destabilize the PAM-binding cleft, perturb REC3 dynamics, and disrupt allosteric coupling to HNH. Together, these findings establish that PAM recognition requires local stabilization, distal coupling, and entropic tuning, rather than a simple consequence of base-specific contacts. This framework provides guiding principles for engineering Cas9 variants with expanded PAM compatibility and improved editing efficiency.
Breast cancer patients participating in a prospective randomized clinical trial who were less than or equal to 49 years of age, had positive axillary nodes, and who received prolonged 1-phenylalanine mustard (L-PAM) as an adjuvant to mastectomy continue (after 4 years) to demonstrate a significantly greater disease-free survival (p = .007) than do patients who received placebo. Benefit was achieved in patients who were less than or equal to 39 years as well as those who were 40-49 years of age. Those in the younger age group showed a greater improvement in disease-free survival at 4 years relative to their controls (32% vs. 69%; p = .01) than did those in the older age group (48% vs. 61%; p = .09). When patients were examined relative to their nodal status, a highly favorable effect was found to have been achieved with L-PAM in those with 1-3 positive nodes (54% vs. 86%; p = .006). Results indicate that both age groups were benefited. When considered over time, they demonstrate that a relatively greater effect was achieved in the younger women. While L-PAM failed to significantly alter the disease-free survival of those with greater than or equal to 4 positive nodes a slightly better effect was achieved in the group less than or equal to 39 years. Since adjuvant chemotherapy has been found to be more effective in premenopausal than postmenopausal women, it has been presumed that decreased ovarian function, as a result of the chemotherapy, is responsible for the findings. To support or repudiate that concept, information regarding serum levels of follicle stimulating hormone (FSH), luteinizing hormone (LH) and estradiol (E2), as well as menstrual function, has been obtained from women receiving L-PAM or L-PAM plus 5-FU therapy. In contrast to findings relative to disease-free survival, ovarian function and menses were most affected in patients 40-49 years of age. Amenorrhea occurred in 73% of patients in that age group and in only 22% of those less than or equal to 39 years (p less than .001). Similarly, a significant increase in LH and FSH and a decrease in E2, all indicative of ovarian suppression, was observed only in the older group of patients. Thus, it is concluded that while ovarian suppression may account for some of the adjuvant chemotherapeutic effect in premenopausal women, the dichotomy of findings in younger and older premenopausal women relative to therapeutic response and ovarian function indicates that other factors could be responsible.
The fine structure of the subcutaneous lymphatic vessel taken from the dorsum pedis of the human adult was investigated without and after impregnation by periodic acid methenamine silver (PAM). To check the stainability of another tissue, the renal glomerulus of the rat was also examined in the same way as outlined above. The membrane system of various cells stains positive. No special surface coat of the endothelial cell of the lymphatic vessel was detected. The fine intracellular filament as well as a continuous basal lamina of the endothelial cell were almost PAM-negative, although a part of the basal lamina was occasionally weakly positive. The chemical composition of the basal lamina of the lymphatic vessel is considered to differ from that of the glomerular blood capillary of the kidney, which is PAM-positive. This difference is thought to be due to the smaller polysaccharide content in the former than in the latter.
RNA-guided CRISPR-derived base editors (BEs) have revolutionized genome editing by enabling targeted base substitutions. However, their application is frequently constrained by the stringent requirement for PAM sequences and low editing precision (bystander editing). Here, we present a robust strategy to overcome these limitations by coupling SpRY, a near-PAM-less Cas9 variant, with truncated CDA1 cytidine deaminases. While this combination enables precise editing of virtually any cytosine in the genome, it initially exhibited suboptimal efficiency. To address this, we systematically screened a diverse panel of candidate DNA-binding proteins and identified that the synergistic fusion of HMGN1 and VP64 substantially enhances editing activity without compromising precision. Importantly, this enhanced editing efficiency was achieved without markedly increasing off-target effects. Our new BEs demonstrated robust performance not only in yeast but also in rice, suggesting broad applicability in gene therapy, precision breeding, and fundamental research.
The ePE-P3-SpRY system enables PAM-flexible prime editing in rice.
In a phase II study of combined adriamycin, L-PAM, and methotrexate with citrovorum factor rescue, 14 of 15 patients with primary advanced or recurrent ovarian malignant tumors obtained an objective remission. In remission, all patients improved their quality of life. So far the median duration of remission is 5+ months for the complete responders and 7 months for the partial responders. Myelosuppression of varying severity occurred in 93% of the courses. The regimen proved highly effective, although toxic in some advanced patients.
Contrary to previous reports, no evidence for a multiple sclerosis-associated agent (MSAA) was obtained when coded brain samples from multiple sclerosis patients and controls were tested in PAM cells.
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Eighty patients with advanced ovarian adenocarcinoma were treated in a prospective, randomized trial comparing a four-drug combination--hexamethylmelamine, cyclophosphamide, methotrexate and 5-fluorouracil--with the oral alkylating agent, melphalan. Treatment with the four-drug combination was associated with a significantly increased overall response rate (75 vs. 54 per cent) (P less than 0.05), more complete remissions (33 vs. 16 per cent) and longer median survival (29 vs. 17 months) (P less than 0.02) but more severe toxicity than occurred with melphalan. Patients with minimal residual disease had a significantly higher overall response rate than patients with extensive residual disease (84 vs. 53 per cent) (P less than 0.05). Patients with advanced disease who achieved a complete remission documented by peritoneoscopy or laparotomy (or both) have a median survival that will exceed three years. The four-drug regimen is more effective than melphalan in the management of advanced ovarian adenocarcinoma.
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BACKGROUND/AIMS: Combination therapy comprising a proton pump inhibitor (PPI), amoxicillin, and metronidazole (PAM) is used to treat clarithromycin-resistant Helicobacter pylori in the Republic of Korea, but eradication rates are decreasing due to an increasing incidence of clarithromycin resistance. We compared PAM, bismuth compounds plus PAM (PAM-B), and the combination of PPI, bismuth compounds, metronidazole, and tetracycline (PBMT) to determine whether PAM-B can achieve an eradication rate higher than that of PAM and comparable to that of PBMT. METHODS: This prospective multicenter study enrolled patients with clarithromycin-resistant H. pylori infections in the Busan and Gyeongsangnam-do of the Republic of Korea between December 2022 and February 2024. RESULTS: In the intention-to-treat (ITT) analysis, the eradication rate was significantly lower in the PAM group (68.2%) than in the PAM-B group (84.8%, p=0.024), whereas the difference from the PBMT group (81.8%) was not significant (p=0.070). The rate remained lowest in the PAM group (75.4%) in the per-protocol (PP) analysis (PAM-B, 96.5%, p=0.001; PBMT, 94.6%, p=0.004). Eradication rates were comparable between the PAM-B and PBMT groups in both the ITT (p=0.640) and PP analyses (p=0.633). Nausea and vomiting were significantly less frequent in the PAM-B group than in the PBMT group (6.8% vs 25.0%; p=0.007). Severe adverse events were rare, and all symptoms resolved after treatment discontinuation. CONCLUSIONS: PAM-B yielded eradication rates higher than those of PAM and comparable to those of PBMT, with no significant increase in the incidence of adverse events, suggesting the potential of PAM-B as a first-line treatment for clarithromycin-resistant H. pylori infection. Further large-scale studies are needed to validate these results. Registered retrospectively with the Clinical Research Information Service (CRIS; KCT0012265).
Mucosal hypertonicity, metabolic inhibitors, or absence of glucose and oxygen enhance mucosal-to-serosal influx of the cationic drug, pralidoxime (PAM), into sacs of everted rat jejunum in vitro. Conversely, efflux of PAM, which is twice the influx rate, is inhibited by mucosal hypertonicity or cyanide and iodoacetate. When sacs containing PAM, 0.87 mM, and glucose, 10 mM, were placed in identical drug- and sugar-containing mediums, the inside (serosal) concentration of PAM fell by over half in 120 min, whereas that of glucose more than doubled. Mucosal hypertonicity depressed PAM efflux and glucose influx regardless of serosal osmolarity. Although azide and mucosal hypertonicity each depressed glucose uptake and oxygen consumption while accelerating net PAM influx, azide more effectively depressed glucose and oxygen uptake, whereas hypertonicity caused greater acceleration of PAM uptake. Hypertonicity did not affect PAM binding to intestinal tissue. Varying mucosal pH did not change PAM or glucose uptake. Thus, mucosal hypertonicity apparently enhances net mucosal-to-serosal transfer of PAM by blocking its active secretion from serosa to mucosa.
Three vinyl monomers, M-1, M-3, and M-5, in which L-phenylalanine p-nitroanilide was acylated with CH2==CHCONH(CH2)nCO--(n = 1, 3, 5) were synthesized. They were co-polymerized with a large excess of acrylamide (co-polymers PAm-1, PAm-3, and PAm-5) and with a large excess of acrylic acid (co-polymers PAc=1, PAc-3, and PCc-5). In addition, M-5 was co-polymerized with acrylamide containing 2.8 mol % of the hydrophobic monomer N-acrylyl-1-naphthylamine (co-polymer PAm-5N). The rates of the chymotrypsin-catalyzed hydrolysis of the nitroanilide groups of M-5 and the various co-polymers were determined over a range of pH. For some of the systems data were also obtained over a range of substrate concentrations to derive values for Vmax and Km. Results obtained with PAm-5 were found to be independent of the chain length of the co-polymer. At pH 7, 25 degrees and with 2.7 X 10(-6) M enzyme, Vmax values for M-5, PAm-k, PAm-5N, and PAc-5 were 5.5, 5.5, 10, and 3.6 X 10(-8) M/S, while Km values were 8.5, 16.5, 10, and 2.2 X 10(-5),respectively, With PAc-5, the pH activity profile was shifted to higher acidities as compared to the profiles obtained with M-5 and PAm-5. The susceptibility of the co-polymers to chymotrypsin attack decreases sharply with a decreasing spacing of the L-phenylalanine p-nitroanilide residue from the backbone of the polymer chains.
Positive allosteric modulators (PAMs) of the delta-opioid receptor (DOR) enhance endogenous opioid signaling while avoiding the convulsant liability of orthosteric agonists. However, the prototypical DOR-PAM, BMS-986187, also potentiates mu-opioid receptor (MOR) signaling, raising concerns regarding respiratory depression and abuse liability. Here, we report a structure-activity study of the benzyloxy moiety of BMS-986187 to improve selectivity for DOR over MOR, while retaining DOR-PAM potency. Fifty-two new analogues and 12 previously reported ones featuring mono- and disubstitution of the benzyl ring and phenyl-heterocycle replacements were synthesized and evaluated in β-arrestin2 recruitment assays. Ortho-substituted derivatives consistently enhanced DOR-PAM potency, although often increased MOR-PAM activity. One pyridyl derivative (compound 35) retained high DOR-PAM potency and efficacy (EC50 = 0.1 μM, Emax = 91%) with no detectable MOR activity. In mice, compound 35 enhanced DOR-mediated reversal of nitroglycerin-induced hyperalgesia, an effect absent in DOR-knockout mice, without enhancing MOR-mediated antinociception, demonstrating in vivo selectivity.
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 has been widely utilized for plant genome editing, but the protospacer adjacent motif (PAM) requirement limits its editing scope. CRISPR/Cas12i3 belongs to the type-VI Cas system that has gained extensive attention due to its smaller size and less restricted canonical TTN PAM sequence. In this study, we explored the newly developed Cas-SF01 system (Cas12i3 variant) for genome editing in oilseed rape. We established an efficient protoplast transformation system in oilseed rape to compare editing efficiency between Cas-SF01 and Cas9. Cas-SF01 shows cleavage activities at the tested 5'-TTN-3' PAM sites with editing outcomes sharing considerable similarities with the CRISPR-Cas9 system in protoplast. Cas-SF01 also induces high efficiency mutagenesis for multiple target sites in stable transformed oilseed rape lines, generating mutants with multilocular silique and male sterile phenotypes. Furthermore, Cas-SF01-derived cytosine base editors (CBEs) were developed to produce targeted C-to-T base edits. Compared to SpCas9, Cas-SF01 has an expanded PAM range and effectively recognizes TTN PAMs, which has substantially broadened the scope of editable sites within the rapeseed genome. No mutations were identified at the putative off-target sites among the edited plants. This study developed a robust, first-of-its-kind Cas12 system in the allotetraploid Brassica napus, expanding the scope of editing and enriching genome-editing toolkits for biological research and genetic improvement.
The distribution of L-phenylalanine mustard (L-PAM) was studied in dogs and mice by high-pressure liquid chromatography. Separation of L-PAM from its products of hydrolysis was accomplished with a mu-Bondapak C18 column, a solvent system composed of 2-methoxyethanol/0.1% acetic acid, and solvent programming with a step gradient. Complete separation was effected in less than 15 min. The half-life for disappearance of L-PAM from mouse blood was 41 min, whereas that from dog blood was 29 min. The monohydroxy derivative of L-PAM, L-MOH, disappeared from dog serum with a half-life of 32 min. L-MOH was not detectable in mouse tissue other than blood at times greater than 15 min after injection. In the dog at 4 hr after injection, the tissue/serum concentration ratios were greater than 1 for liver, spleen, intestine, skeletal muscle, urinary bladder and gallbladder. The concentration of L-PAM in the bile was approximately 500 times higher than that in serum.