The aim of this investigation was to determine the optimal storage medium for testicular hypothermic transportation and identify the ideal concentration for the application of the protective agent 5-aminolevulinic acid (5-ALA). Furthermore, this study aimed to explore the underlying mechanism of the protective effects of 5-ALA. First, we collected and stored mouse testicular fragments in different media, including Hank's balanced salt solution (HBSS; n = 5), Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12; n = 5), and alpha-minimum essential medium (αMEM; n = 5). Storage of testicular tissue in HBSS preserved the integrity of testicular morphology better than that in the DMEM/F12 group ( P < 0.05) and the αMEM group ( P < 0.01). Testicular fragments were subsequently placed in HBSS with various concentrations of 5-ALA (0 [control], 1 mmol l -1 , 2 mmol l -1 , and 5 mmol l -1 ) to determine the most effective concentration of 5-ALA. The 2 mmol l -1 5-ALA group ( n = 3) presented the highest positive rate of spermatogonial stem cells compared with those in the control, 1 mmol l -1 , and 5 mmol l -1 5-ALA groups. Finally, the tissue fragments were preserved in HBSS with control ( n = 3) and 2 mmol l -1 5-ALA ( n = 3) under low-temperature conditions. A comparative analysis was performed against fresh testes ( n = 3) to elucidate the underlying mechanism of 5-ALA. Gene set enrichment analysis (GSEA) for WikiPathways revealed that the p38 mitogen-activated protein kinase (MAPK) signaling pathway was downregulated in the 2 mmol l -1 5-ALA group compared with that in the control group (normalized enrichment score [NES] = -1.57, false discovery rate [FDR] = 0.229, and P = 0.019). In conclusion, these data suggest that using 2 mmol l -1 5-ALA in HBSS effectively protected the viability of spermatogonial stem cells upon hypothermic transportation.
Male ; Animals ; Testis/cytology* ; Aminolevulinic Acid/pharmacology* ; Mice ; Organ Preservation/methods* ; Organ Preservation Solutions/pharmacology* ; Cryopreservation/methods*

OBJECTIVE: To explore whether microneedle pretreatment can significantly improve the efficacy and safety of 5-aminolevulinic acid (ALA)-photodynamic therapy (PDT) in the treatment of oral leukoplakia. METHODS: A non-randomized controlled clinical trial was conducted. Patients with clinical and pathological diagnosis of oral leukoplakia in the Department of Oral Mucosa, Peking University School and Hospital of Stomatology were divided into experimental group and control group. The control group was treated with conventional ALA-PDT, and the experimental group was pretreated with micro- needle buckling under superficial anesthesia with lidocaine before conventional ALA-PDT. The clinical manifestations of the two groups were recorded, the lesion area was measured, the clinical efficacy was evaluated, the number of treatment sessions and treatment unit duration were analyzed, and the pain after treatment was evaluated by visual analogue scale. The above data of the two groups were statistically analyzed. RESULTS: A total of 11 patients were included in the experimental group and 19 patients were included in the control group. The complete remission rate of the experimental group and the control group was 45.5% and 36.8%, the partial remission rate was 54.5% and 57.9%, and the no remission rate was 0% and 5%, respectively. There was no significant difference in the treatment effect between the two groups. Meanwhile, the treatment unit duration of the experimental group and the control group were (9.05±5.74) min/cm² and (21.38±15.44) min/cm², respectively, and the number of treatment sessions were (2.36±0.67) times and (3.58±1.57) times, respectively. These differences between the two groups were statistically significant (t=-3.125, P < 0.05; t=-2.932, P < 0.05). Similarly, multiple linear regression analysis with 7 factors including age, dysplastic pathology, lesion classification, etc., also confirmed that pretreatment could significantly shorten the treatment unit duration (P < 0.05). In addition, there was no significant difference in pain score (visual analogue scale) between the two groups after treatment, and the microneedle puncture pretreatment did not increase the adverse reactions of ALA-PDT treatment. CONCLUSION Microneedle pretreatment followed by conventional ALA-PDT shows a good clinical effect on oral leukoplakia, which can significantly shorten the clinical treatment time, reduce the number of visits, and save medical costs.
Humans ; Photochemotherapy/instrumentation* ; Leukoplakia, Oral/drug therapy* ; Aminolevulinic Acid/therapeutic use* ; Male ; Female ; Middle Aged ; Adult ; Needles ; Photosensitizing Agents/therapeutic use* ; Aged ; Combined Modality Therapy

Basal cell carcinoma (BCC) and cutaneous squamous cell carcinoma (cSCC), as certain forms of non-melanoma skin cancer (NMSC) or keratinocyte carcinoma, are the most common forms of malignant neoplasms worldwide (Sharp et al., 2024). BCC and cSCC have been identified as two major components of NMSC, comprising one-third of all malignancies (Burton et al., 2016). Generally speaking, patients with NMSC tend to have relatively favorable survival outcomes, while different histopathological subtypes of NMSC exhibit distinct biological behaviors (Stătescu et al., 2023). Keratinocyte carcinoma, although not considered as deadly as melanoma, tends to metastasize if left untreated (Civantos et al., 2023; Nanz et al., 2024). cSCC can evolve locally, then aggressively metastasize, invade, and even lead to fatal consequences in a subset of patients (Winge et al., 2023). A solid, pigmented, smooth plaque or a hyperkeratotic papule with or without central ulceration and hemorrhage appears to be characteristic of cSCC (Thompson et al., 2016; Zhou et al., 2023). Of note, a rare type of intraepidermal cSCC in situ often appears as a velvety, demarcated, slightly raised erythematous plaque on the genitalia of men (Yamaguchi et al., 2016). Accounting for approximately 16.0% of scalp tumors and with a rising incidence, cSCC is now the second most common NMSC in humans (Verdaguer-Faja et al., 2024). According to the latest statistics, up to 2%‒5% of cSCCs in situ may gradually progress into invasive cSCCs in the final step (Rentroia-Pacheco et al., 2023). Several risk factors for the carcinogenesis and development of cSCC have been identified, including age, accumulative exposure to ultraviolet light radiation A and B, human papillomavirus infection, arsenic ingestion, chronic scarring, xeroderma pigmentosa, a relevant history of ionizing radiation, androgenetic alopecia in males, and immunosuppression therapy (Martinez and Otley, 2001; Welsch et al., 2012; Mortaja and Demehri, 2023).
Humans ; Aminolevulinic Acid/therapeutic use* ; Skin Neoplasms/pathology* ; Photochemotherapy/methods* ; Female ; Carcinoma, Squamous Cell/pathology* ; Middle Aged ; Photosensitizing Agents/therapeutic use* ; Carcinoma, Basal Cell/drug therapy*

The probiotic strain Escherichia coli Nissle 1917 (EcN) with high biocompatibility and susceptibility to genetic modification is often applied in bacterial therapies for cancer. However, most studies have used plasmids as vectors to construct engineering strains from EcN. Plasmid-based expression systems suffer from genetic instability, and they need antibiotic selective pressure to maintain high copy number. This study aimed to employ EcN for synthesizing the photosensitizer 5-aminolevulinic acid (5-ALA). Firstly, the key genes of 5-ALA synthesis, hemA^M and hemL, were integrated into the EcN genome by the phage integration technique. Then, chemically inducible chromosomal evolution (CIChE) was adopted to increase the copy number of hemA^M and hemL and thus improved the stable synthesis of 5-ALA. The in vitro cell experiments verified that the constructed engineering strain can deliver stably synthesized 5-ALA to tumor cells and inhibit their growth. This study provided a basis for applying the engineering strains of EcN in the photodynamic therapy for tumors.
Escherichia coli/metabolism* ; Aminolevulinic Acid/metabolism* ; Photosensitizing Agents/pharmacology* ; Plasmids/genetics* ; Chromosomes, Bacterial/genetics* ; Genetic Engineering ; Humans ; Probiotics ; Photochemotherapy

Heme, which exists widely in living organisms, is a porphyrin compound with a variety of physiological functions. Bacillus amyloliquefaciens is an important industrial strain with the characteristics of easy cultivation and strong ability for expression and secretion of proteins. In order to screen the optimal starting strain for heme synthesis, the laboratory preserved strains were screened with and without addition of 5-aminolevulinic acid (ALA). There was no significant difference in the heme production of strains BA, BAΔ6 and BAΔ6ΔsigF. However, upon addition of ALA, the heme titer and specific heme production of strain BAΔ6ΔsigF were the highest, reaching 200.77 μmol/L and 615.70 μmol/(L·g DCW), respectively. Subsequently, the hemX gene (encoding the cytochrome assembly protein HemX) of strain BAΔ6ΔsigF was knocked out to explore its role in heme synthesis. It was found that the fermentation broth of the knockout strain turned red, while the growth was not significantly affected. The highest ALA concentration in flask fermentation reached 82.13 mg/L at 12 h, which was slightly higher than that of the control 75.11 mg/L. When ALA was not added, the heme titer and specific heme production were 1.99 times and 1.45 times that of the control, respectively. After adding ALA, the heme titer and specific heme production were 2.08 times and 1.72 times higher than that of the control, respectively. Real-time quantitative fluorescent PCR showed that the expressions of hemA, hemL, hemB, hemC, hemD, and hemQ genes at transcription level were up-regulated. We demonstrated that deletion of hemX gene can improve the production of heme, which may facilitate future development of heme-producing strain.
Gene Deletion ; Bacillus amyloliquefaciens/metabolism* ; Aminolevulinic Acid/metabolism* ; Heme/metabolism* ; Fermentation

5-aminolevulinic acid (5-ALA) plays an important role in the fields of medicine and agriculture. 5-ALA can be produced by engineered Escherichia coli and Corynebacterium glutamicum. We systematically engineered the C4 metabolic pathway of C. glutamicum to further improve its ability to produce 5-ALA. Firstly, the hemA gene encoding 5-ALA synthase (ALAS) from Rhodobacter capsulatus and Rhodopseudomonas palustris were heterologously expressed in C. glutamicum, respectively. The RphemA gene of R. palustris which showed relatively high enzyme activity was selected. Screening of the optimal ribosome binding site sequence RBS5 significantly increased the activity of RphemA. The ALAS activity of the recombinant strain reached (221.87±3.10) U/mg and 5-ALA production increased by 14.3%. Subsequently, knocking out genes encoding α-ketoglutarate dehydrogenase inhibitor protein (odhI) and succinate dehydrogenase (sdhA) increased the flux of succinyl CoA towards the production of 5-ALA. Moreover, inhibiting the expression of hemB by means of sRNA reduced the degradation of 5-ALA, while overexpressing the cysteine/O-acetylserine transporter eamA increased the output efficiency of intracellular 5-ALA. Shake flask fermentation using the engineered strain C. glutamicum 13032/∆odhI/∆sdhA-sRNAhemB- RBS5RphemA-eamA resulted in a yield of 11.90 g/L, which was 57% higher than that of the original strain. Fed-batch fermentation using the engineered strain in a 5 L fermenter produced 25.05 g/L of 5-ALA within 48 h, which is the highest reported-to-date yield of 5-ALA from glucose.
Aminolevulinic Acid/metabolism* ; Corynebacterium glutamicum/metabolism* ; Fermentation ; Metabolic Engineering ; Rhodobacter capsulatus/enzymology* ; Rhodopseudomonas/enzymology*

To investigate the effect of 5-aminolevulinic acid photodynamic therapy (ALA-PDT) on () biofilm. biofilms were constructed on a cell slide and treated with ALA-PDT.According to different light doses,the biofilms were divided into six groups:ALA-PDT group [ALA-PDT1 (50 J/cm),ALA-PDT2 group (100 J/cm),ALA-PDT3 group (200 J/cm)],ALA-only group (ALA group),light-only group (LED),and a negative control group (ALA-PDT-group).The biofilm structure and the ratio of the dead bacteria/live bacteria were observed using a laser confocal microscope (CLSM).Biofilm viability was measured using the XTT assay. CLSM showed that the biofilm structures of ALA group and LED group were not significantly different from that of ALA-PDT-group,whereas the biofilm structure was more seriously damaged in ALA-PDT1 group,ALA-PDT2 group,and ALA-PDT3 group than in the ALA-PDT-group.The ratios of the dead/live bacteria in ALA-PDT-group,ALA group,LED group,ALA-PDT1 group,ALA-PDT2 group,and ALA-PDT3 group were 0.350±0.033, 0.305±0.046, 0.330±0.032, 1.525±0.439, 2.293±0.148 and 3.092±0.189,respectively.ALA group(=0.003, =1.000)and LED group(=-0.025, =1.000)did not significantly differ from the ALA-PDT-group.However,the ratio of dead/live bacteria in ALA-PDT-group was significantly lower than those in ALA-PDT1 group (=-0.162, <0.001),ALA-PDT2 group (=-0.254, <0.001),and ALA-PDT3 group (=-0.352, <0.001).The values of the XTT assay were were 0.462±0.028,0.465±0.044,0.437±0.047,0.301±0.040,0.207±0.001,and 0.110±0.007,respectively,in ALA-PDT-group,ALA group,LED group,ALA-PDT1 group,ALA-PDT2 group,and ALA-PDT3 group.Although the values of XTT assay in ALA(=-0.044, =1.000)and LED groups (=-0.020, =1.000)did not significantly differ from that in ALA-PDT-group,it was significantly higher in ALA-PDT-group than in ALA-PDT1 group (=1.175, <0.001),ALA-PDT2 group (=1.942, <0.001),and ALA-PDT3 group (=-0.352, =2.742, <0.001). ALA-PDT has an inhibitory effect on biofilm.ALA-PDT destroys biofilm structure and inhibits biofilm viability.
Aminolevulinic Acid ; Biofilms ; Photochemotherapy ; Photosensitizing Agents ; Propionibacterium acnes

With recent developments in photosensitizers and light delivery systems, topical 5-aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) has become the fourth alternative therapeutic approach in the management of oral leucoplakia (OLK) due to its minimally invasive nature, efficacy, and low risk of systemic side effects and disfigurement. This report presents step-by-step guidelines for applying topical ALA-PDT in the management of OLK based on both the clinical experience of the authors and a systematic review of the current literature. Studies using protocols with standardized parameters and randomized clinical trials at multiple centres with adequate sample sizes and both interim and long-term follow-ups are needed before universally applicable guidelines can be produced in this field.
Aminolevulinic Acid ; administration & dosage ; therapeutic use ; Humans ; Leukoplakia, Oral ; therapy ; Photochemotherapy ; methods ; Photosensitizing Agents ; administration & dosage ; therapeutic use ; Practice Guidelines as Topic

STUDY DESIGN: A retrospective study. PURPOSE: We report our experience with 5-aminolevulinic acid (5-ALA)–assisted resection of spinal cord ependymomas in adults. OVERVIEW OF LITERATURE: Ependymoma is the most frequent primary spinal cord tumor in adults. Surgery is the treatment of choice in most cases. However, while complete resection is achieved in approximately 80% of cases, clinical improvement is achieved in 15% only. Five-ALA fluorescence–guided surgery seems to be useful for this tumor type. METHODS: We studied 14 patients undergoing 5-ALA fluorescence-guided surgery for spinal cord ependymomas in our service. The modified McCormick classification was used to determine clinical status and the degree of resection was assessed with magnetic resonance imaging. RESULTS: Of the 14 patients, the tumor showed an intense emission of fluorescence in 12 and the fluorescence was weak and nonuniform in two. Complete resection was achieved in 11 cases. According to the McCormick classification, 10 patients improved, two remained the same, and two deteriorated. CONCLUSIONS: Our results confirm that 5-ALA fluorescence-guided resection is useful in spinal cord ependymoma resection. Although the rate of complete resections is similar to that in published series without 5-ALA, clinical results are better when using 5-ALA with a lower percentage of clinical deterioration.
Adult ; Aminolevulinic Acid ; Classification ; Ependymoma ; Fluorescence ; Humans ; Magnetic Resonance Imaging ; Retrospective Studies ; Spinal Cord Neoplasms ; Spinal Cord

OBJECTIVETo evaluate the photodynamic therapy (PDT) against multi-antibiotic-resistant Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S.epidermidis) obtained from patients with chronic rhinosinusitis (CRS).

METHODSForty-five CRS patients who had been given medical treatment but still needed endoscopic surgery were included in this study. The mucus from middle meatus was collected from these patients during surgery, followed by separation of S. aureus and S. epidermidis and drug sensitive test. The strains which could form biofilm were selected. Light emitting diode (LED) array with a major wavelength of (633±10) nm was used as light source and 5-Aminolevulinic acid (ALA) was used as photosensitizer in this PDT experiment. The safe range of LED dose and ALA concentration which were not toxic to bacteria by themselves were confirmed, and then did PDT experiment on S. aureus and S. epidermidis. The data of bacterial colony forming unit were transformed to lgCFU before statistical analysis.The Graph Pad Prism 5 software was used to analyzed the data.

RESULTSThirteen S. aureus and 16 S. epidermidis were included in this experiment(from 45 patients), all of them were multi-antibiotic-resistant bacteria, and four of S. aureus and five of S. epidermidis could form biofilm in each group. In planktonic S. aureus experiment, the mean lgCFU was 8.32±0.31 in control group whereas the experiment group was 6.47±0.67 (t=9.01, P<0.01), and in planktonic S. epidermidis experiment the final data was 8.34±0.20 (control group) and 6.97±0.59 (experiment group) (t=8.84, P<0.01). In biofilm S. aureus experiment, the mean lgCFU was 8.68±0.05 (control group), 6.90±0.96(experiment group) (t=3.68, P<0.05); and in biofilm S. epidermidis experiment the data was 8.67±0.05 (control group), 7.29±0.61 (experiment group, t=5.07, P<0.01).

CONCLUSIONOur results demonstrated that ALA-mediated PDT on multi-antibiotic-resistant S. aureus and S. epidermidis from CRS patients was effective in vitro. Additional work defining if the PDT treatment would damage the nasal mucosa and further checking the effectiveness of PDT in vivo is still needed.

Aminolevulinic Acid ; therapeutic use ; Anti-Bacterial Agents ; Biofilms ; Drug Resistance, Multiple, Bacterial ; Humans ; Light ; Photochemotherapy ; Photosensitizing Agents ; therapeutic use ; Rhinitis ; drug therapy ; microbiology ; Sinusitis ; drug therapy ; microbiology ; Staphylococcal Infections ; complications ; drug therapy ; Staphylococcus